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HIV TYPE 1 GENETIC DIVERSITY IN SELECTED COUNTIES

OF KENYA

KITAWI ROSE CHARI

MASTER OF SCIENCE
(Epidemiology)

JOMO KENYATTA UNIVERSITY OF


AGRICULTURE AND TECHNOLOGY

2016
HIV Type 1 Genetic Diversity In Selected Counties Of Kenya

Rose Chari Kitawi

A thesis submitted in partial fulfillment for the Degree of Master of Science in


Epidemiology in the Jomo Kenyatta University of Agriculture and Technology

2016
DECLARATION
This thesis is my original work and has not been presented for a degree at any other university.

Signature…………………………………. Date………………………..

Rose Chari Kitawi

This thesis has been submitted to the University for Examination with our approval as
supervisors:
Signature…………………………………. Date……….
Prof. Washingtone Ochieng’
KEMRI, KENYA

Signature…………………………………. Date………………………..
Prof. Anne W. T. Muigai,
JKUAT, KENYA

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DEDICATION

To my loving parents who have sacrificed so much for me to reach where I am.

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ACKNOWLEDGEMENT
I am really grateful for the „help from above‟. What would I do without You?
I am grateful to my parents, my brothers and my sisters for being there for me whenever I needed
them. Thanks so much for your support.
I acknowledge the help of Professor Washingtone Ochieng and Professor Anne Muigai, my
supervisors, for valuable guidance and input during my work and thesis write-up.
This research project would have come to nothing were it not for the support and valuable input
of my colleagues Timothy, Maureen, Ruth, Geoffrey, Javan, Nancy, Joyceline, Lucy and all the
KEMRI CVR staff who have really been resourceful in this project.
I am grateful to the patients who consented to having the study done on them. Without you, the
project would not even have begun.

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TABLE OF CONTENTS

DECLARATION .......................................................................................................................... ii

DEDICATION ............................................................................................................................. iii

ACKNOWLEDGEMENT........................................................................................................... iv

LIST OF FIGURES ..................................................................................................................... ix

LIST OF TABLES ........................................................................................................................ x

LIST OF APPENDICES ............................................................................................................. xi

LIST OFABBREVIATIONS ..................................................................................................... xii

DEFINITION OF OPERATIONAL TERMS .......................................................................... xv

ABSTRACT................................................................................................................................ xvi

CHAPTER ONE ........................................................................................................................... 1

INTRODUCTION ........................................................................................................................ 1

1.1Background Information ......................................................................................................................1

1.2 Statement of the Problem ....................................................................................................................3

1.3 Justification of the Study .....................................................................................................................4

1.4 Research Questions .............................................................................................................................5

1.5 Objectives ............................................................................................................................................5

1.5.1 General Objective ......................................................................................................................... 5


1.5.2 Specific Objectives ....................................................................................................................... 5
CHAPTER TWO .......................................................................................................................... 6

LITERATURE REVIEW ............................................................................................................ 6

2.1 The HIV-1 Structure and Genome Organization.................................................................................6

2.2 HIV-1 Diversity...................................................................................................................................8

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2.3 Epidemiology of HIV strains ..............................................................................................................9

2.4 The global HIV-1 genomic database and phylogenetic profiles .......................................................12

2.5 Kenyan HIV-1 Genetic Profile ..........................................................................................................13

2.6 Co-receptor Usage .............................................................................................................................16

2.6.1. HIV Phenotypes and Coreceptor Usage .................................................................................... 16


2.6.2 Mechanism of viral entry into host cell and coreceptor switching ............................................. 16
2.7 HIV Envelop Diversity and Potential N-Linked Glycosylation Patterns ..........................................17

CHAPTER THREE .................................................................................................................... 20

MATERIALS AND METHODS ............................................................................................... 20

3.1 Study design ......................................................................................................................................20

3.2 Study site ...........................................................................................................................................20

3.3 Study population ...............................................................................................................................22

3.3.1 Inclusion Criteria ........................................................................................................................ 22


3.3.2 Exclusion criteria ........................................................................................................................ 22
3.4 Sampling............................................................................................................................................22

3.4.1 Sample Size Determination ........................................................................................................ 22


3.4.2 Sampling Procedure ................................................................................................................... 23
3.5 Ethical Considerations .......................................................................................................................23

3.6 Laboratory and Analytical Procedures ..............................................................................................23

3.6.1 Sample processing ...................................................................................................................... 23


3.6.2 Viral Load Determination .......................................................................................................... 24
3.6.3 Nucleic Acid Preparation ........................................................................................................... 24
3.6.3.1 DNA Extraction Procedure.................................................................................................. 24
3.6.3.2 RNA Extraction Procedure .................................................................................................. 26
3.6.4 Choice of Primers for Polymerase Chain Reaction (PCR) ......................................................... 27
3.6.5 Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Round 1 PCR for RNA
Samples ............................................................................................................................................... 28
3.6.6 Nested PCR for RNA Samples ................................................................................................... 29
3.6.7 DNA PCR ................................................................................................................................... 29

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3.6.8 Gel Electrophoresis .................................................................................................................... 30
3.6.9 Purification and Sequencing ....................................................................................................... 31
3.6.10: HIV-1 Subtyping and Subtype Analysis ................................................................................. 31
3.6.11: Determination of Coreceptor Usage ........................................................................................ 32
3.6.12: Determination of envelope protein diversity ........................................................................... 33
3.7 Data Management and Analysis ........................................................................................................34

CHAPTER FOUR....................................................................................................................... 35

RESULTS..................................................................................................................................... 35

4.1 Patient Characteristics .......................................................................................................................35

4.2 HIV-1 subtyping ................................................................................................................................39

4.2.1 Phylogenetic Analysis ................................................................................................................ 39


4.2.1.1. Phylogenetic analysis for the envelope C2V3 Sequences .................................................. 39
4.2.1.2 Phylogenetic Analysis of the Pol RT sequences ................................................................. 41
4.2.2 Comparative sensitivities of common genotyping tools used for HIV-1 subtyping .................. 43
4.2.3 Genetic Heterogeneity of HIV-1 Pol RT gene from different blood compartments .................. 45
4.2.4 Genetic heterogeneity of HIV-1 C2V3 envelope gene from different blood compartments ...... 46
4.2.5 Comparative distribution of HIV genetic variants between blood compartments...................... 47
4.2.6 Subtype Distribution by County ................................................................................................. 48
4.3 Co-receptor Usage .............................................................................................................................50

4.4 Patterns of Potential N-Linked Glycosylation Sites (PNGs) .............................................................52

CHAPTER 5 ................................................................................................................................ 58

DISCUSSION .............................................................................................................................. 58

5.1 Patient Characteristics .......................................................................................................................58

5.2 HIV-1 subtype diversity ....................................................................................................................59

5.3 Co-receptor Usage .............................................................................................................................62

5.4 Number of Potential N-Linked Glycosylation (PNG) Sites ..............................................................63

5.5 Conclusion .........................................................................................................................................64

5.6 Limitations of the study.....................................................................................................................65

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5.7 Recommendations .............................................................................................................................65

REFERENCES............................................................................................................................ 66

APPENDICES ............................................................................................................................. 79

Appendix 1A: Consent Information for Study Participants (English) ....................................................79

Appendix1B: Fomu ya idhini kwa wanauhusika katika utafiti huu (Kiswahili version) ........................82

Appendix 2A: Parental/Guardian consent for children 13-17 years (English). .......................................85

Appendix 2B: Idhini ya Mzazi wa mtoto mwenye umri wa miaka 13-17 (Kiswahili Version). .............86

Appendix 3A: QUESTIONNAIRE (English version) ............................................................................87

Appendix 3B: MAHOJIANO ..................................................................................................................89

Appendix 4: Analysis of concordance between various subtyping tools ................................................91

Appendix 5: Coreceptor Usage and Number of Potential N-Glycosites .................................................96

Appendix 6: Request for SSC and ERC waiver ......................................................................................99

Appendix 7: ERC Approval ..................................................................................................................100

Appendix 8: SSC Approval ...................................................................................................................101

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LIST OF FIGURES
Figure 2.1: Diagram of mature HIV virion ......................................................................... 7
Figure 2.2: The HIV-1 genome map based on HXB2 reference sequence.
HIV Immunology 2002 ................................................................................... 7
Figure 2.3: Regional distribution of HIV 1 subtypes and recombinants in 2000-2003 (a)
and 2004-2007 (b) ........................................................................................... 11
Figure 2.4: Neighbour joining phylogenetic tree for HIV-1 group M viruses .................... 13
Figure 3.1: Map of Kenya Counties with corresponding HIV prevalence ......................... 21
Figure 4.1: Phylogenetic Relationships of HIV-1 envelope C2V3 gene isolates ................ 40
Figure 4.2: Phylogenetic Relationships of HIV-1 envelope pol RT gene isolates ............... 42
Figure 4.3: Proportion of sequences containing PNGs at specific amino acid positions. ...... 56

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LIST OF TABLES
Table 1.1: Worldwide Distribution of HIV (in Thousands). ............................................................ 2
Table 2.1: Subtype Distribution in Kenya .......................................................................... 15
Table 3.1: Pool of HIV specific primers used for amplification of env and pol RT
regions by polymerase chain reaction (PCR) ..................................................... 28
Table 4.1: Patient characteristics based on pol RT phylogenetic subtypes .......................... 37
Table 4.2: Patient characteristics based on C2V3 phylogenetic subtypes ......................... 38
Table 4.3: Comparative concordance of various subtyping tools for assignment of
HIV-1 envC2V3 subtypes ................................................................................ 44
Table 4.4: Comparison of concordance for various subtyping tools for the pol RT gene .... 45
Table 4.5: Distribution and compartmentalization of HIV-1 Pol-RT subtypes .................... 46
Table 4.6: Distribution and compartmentalization of envelope C2V3 subtypes .................. 47
Table 4.7 Comparative frequency of subtypes by genetic region and compartment .......... 48
Table 4.8: Variability of C2V3 HIV genotypes within a given geographic
locations across Kenya ....................................................................................... 49
Table 4.9: Variability of Pol RT HIV genotypes within a given geographic locations
across Kenya ...................................................................................................... 50
Table 4.10: The distribution by number and (proportions), of viral tropism across
HIV subtype variants ......................................................................................... 52
Table 4.11: The distribution and clustering of specific amino acid PNG sites according to
viral tropism and NA source material .............................................................. 54
Table 4.12: Mean PNGs for 55 isolates by subtype, nucleic acid source and tropism ........ 55

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LIST OF APPENDICES
Appendix 1A: Consent Information for Study Participants (English) ........................ 79
Appendix 1B: Consent Information for Study Participants (Kiswahili) ....................... 82
Appendix 2A: Parental/Guardian Consent for Children 13-17years (English) ............ 85
Appendix 2B: Parental/Guardian Consent for Children 13-17years (Kiswahili) ......... 86
Appendix 3A: Questionnaire (English) ......................................................................... 87
Appendix 3B: Questionnaire (Kiswahili) ...................................................................... 89
Appendix 4: Analysis of concordance of various subtyping tools .............................. 91
Appendix 5: Coreceptor Usage and Number of Potential N-Glycosites...................... 96
Appendix 6: Letter to request waiver for ERC and SSC ............................................ 99
Appendix 7: Letter for ERC Approval ........................................................................ 100
Appendix 8: Letter for SSC Approval ........................................................................ 101

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LIST OFABBREVIATIONS

3TC Lamivudine
ABC Abacavir
AIDS Acquired Immune Deficiency Syndrome
ARV/ART Antiretroviral Therapy
AZT Azidothymidine (Zidovudine)
CCC Comprehensive Care Centre
cDNA complement deoxy ribonucleic acid
COMET COntext-based Modeling for Expeditious Typing
CREATES Centre for Research in Therapeutic Sciences
CRFs Circulating Recombinant Forms
CVR Centre for Virus Research
d4T Stavudine
DDBJ DNA Databank of Japan
DNA Deoxy-ribonucleic Acid
dNTPs Deoxyribonucleotide triphosphate
DRC Democratic Republic of Congo
EDTA Ethylene Diamine Tetraacetic Acid
EFV Efavirenz
EMBL-EBI European Molecular Biology Laboratory‟s European Bioinformatic Institute
ERC Ethical Review Committee
FPR False Positive Rate (used here to mark the cut-off for coreceptor usage).
HAART Highly Active Anitretroviral Therapy
HIV Human Immunodeficiency Virus
HIV-1 Human Immunodeficiency Virus Type 1
JPHMM Jumping Profile Hidden Markov Model
KEMRI Kenya Medical Research Institute
KDHS Kenya Demographic and Health Survey
KNBS Kenya National Bureau of Statistics

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LANL Los Alamos National Laboratory
MEGA Molecular Evolutionary Genetic Analysis
MgCl2 Magnesium Chloride
min Minutes
ml Millilitres
mM Millimolar
NACC National AIDS Control Council
NASCOP National AIDS and STI Control Programme
NCBI National Center for Biotechnology Information
NNRTI Non Nucleoside Reverse Transcriptase Inhibitors
NSI Non-Syncytium Inducing
nt Nucleotide
NVP Nevirapine
PBMCs Peripheral Blood Mononuclear Cells
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PLWHA People Living With HIV AIDS
pmol Picomole
PNGs Potential N-linked Glycosylation Sites
REGA Subtyping tool of the Rega Institute
RNA Ribonucleic Acid
RT Reverse Transcriptase
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
s Seconds
SDR Subtype Diversity Ratio
SI Syncytium Inducing
SOP Standard Operating Procedures
SPSS Statistical Package for Social Sciences
SSC Scientific Steering Committee
TDF Tenofovir disoproxil fumarate (Tenofovir)
URFs Unique Recombinant Forms

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UV Ultra violet
μg micrograms
µl Microlitres

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DEFINITION OF OPERATIONAL TERMS
CCR5 G-protein coupled beta chemokine receptor type 5 in the C-C chemokine group
CXCR4 C-X-C chemokine receptor type 4
HXB2 This HIV virus is the most commonly used reference strain for many different
HIV studies. All structural data related to the envelope region published so far
translates residue numbers into the HXB2 numbering scheme.
NXS Sequon with N referring to Asparagine, X to any amino acid apart from proline
and S to serine
NXT Sequon with N referring to Asparagine, X to any amino acid apart from proline
and T to Threonine
Sequon A sequon is a sequence of consecutive amino acids in a protein that can serve as
the attachment site to a polysaccharide, which is frequently an N-linked glycan.
The polysaccharide is linked to the protein via the nitrogen atom in the side chain
of asparagine (Brooks, Dwek, & Schumacher, 2002).

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ABSTRACT
The human immunodeficiency virus (HIV) is one of the most highly genetically diverse viruses
known. This extensive genetic variation has many implications on anti-retroviral drug response,
viral transmission and vaccine and diagnostics designs and development. A few studies
conducted over the last 10 years have shown that HIV-1A is the predominant subtype circulating
in Kenya, although there is evidence of increasing emergence of subtypes C and D. This cross-
sectional study was conducted in Kisumu and Homabay, Kajiado, Nakuru, Kiambu and Kilifi
Counties as part of a countrywide genetic study of HIV-1 diversity. The aim was to describe
HIV-1 subtype diversity in the designated counties and the tropism and potential N-linked
glycosylation site (PNGs) patterns of the isolates obtained. Five ml of blood was obtained from
consenting HIV-1 positive patients and processed to obtain plasma and cells. The plasma was
used to obtain viral RNA from the free virus and cells were used to obtain viral DNA. The
nucleic material obtained was amplified using polymerase chain reaction (PCR), purified and
then sequenced. The sequences were used to conduct phylogenetic analysis so as to deduce
evolutionary patterns of HIV-1 using distance matrix. Co-receptor usage and potential N-linked
Glycosylation (PNG) patterns of HIV-1 envelop was examined using generic bioinformatics tool.
The results showed that subtype A1 is the predominant subtype (70%) although the percentage of
recombinants is increasing. With up to 30% recombinant strains observed in the pol RT region,
recombination appeared to be highest in the cell-free compartment (in plasma)- 31.8%, than in
cellular compartment– 10.5%. The partial envelope sequences were also examined for coreceptor
usage and the patterns of PNGs. At three False Positive Rate (FPR) cut-off points for CXCR4
usage i.e. 5%, 10% and 20%, the relationship between subtype and viral tropism was found to be
significant (p=0.037; p=0.016 and p=0.005 for FPR of 5%, 10% and 20% respectively) using χ2
test. A significant difference was found between subtype and mean PNGs, using ANOVA, at
specific amino acid positions - N296 (p=0.021), N302 (p=0.034) and N366 (p=0.016) but not for
the specific PNG patterns examined (NXT, NXS and NNX(S)T). Although R5-tropic isolates had
fewer PNGs, on average, than X4-tropic isolates, the difference was not significant (p=0.303).
The trend of R5-tropic isolates having more PNGs than the X4-tropic isolates was observed for
subtype A1 and not the other subtypes. A significant difference was found when PNGs were
examined by nucleic acid source for the NXT pattern (p=0.016), and when total PNGs for all

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patterns was considered (p=0.011); with the DNA isolates having more PNGs or average than the
RNA virus. The PNG pattern results obtained call for more studies to be done on subtype A1 as it
has unique characteristics in the PNG pattern. Continued surveillance of the prevailing subtypes
in the different regions in the country will help monitor the transmission patterns of the virus and
therefore help in coming up with adequate measures to reduce infection rates.

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CHAPTER ONE

INTRODUCTION

1.1Background Information
The UNAIDS 2013 Global Fact Sheet estimates that 35 million people were living with
HIV globally in 2013, of these, 3.2 million were children (UNAIDS, 2014). The greatest
burden of HIV AIDS lies in sub-Saharan Africa, which has 70%, or 24.7 million of the
global population of people living with HIV-AIDS (PLWHA). The region with the least
number of people living with HIV AIDS is the Middle East and North Africa. The
burden of new infections was also highest in Africa (1.5 million) and lowest in the
Carribean (12,000). There were about 1.1 million AIDS related deaths in Sub-Saharan
Africa, the global AIDS related deaths being about 1.5 million. The global regional
epidemiologic statistics of HIV-1 is summarized in Table 1.1. So far four distinct
lineages of HIV-1 have been characterized from sequences accumulated in the past 25-
30 years and include groups M (Main), O (Outlier), N (non-M, non-O or New) and P
(Putative) (Keele et al., 2006; Plantier, Leoz, & Dickerson, 2009). The majority of
global HIV-1 infections are due to Group M viruses which display a tremendous amount
of genetic variability (Worobey, 2007). Nine different (pure) subtypes (subtypes A, B,
C, D, F, G, H, J, and K), over 55 circulating recombinant forms (CRFs) and several
unique recombinant forms (URFs) have been described (Foley et al., 2013). Currently,
in the Los Alamos HIV database, over 70 CRFs have been described. Inter-subtype
recombinant genomes are commonly found in dually infected individuals, while CRFs
arise from HIV-1 recombinant genes that have infected three or more persons who are
not related epidemiologically (R. Lihana, Ssemwanga, Abimiku, & Ndembi, 2012).
Subtypes A and F have additional sub-subtypes A1, A2, A3, A4, A5, F1 and F2 based
on genetic variation of between 15-20% (R. Lihana et al., 2012; Taylor & Hammer,

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2008). Like other genetic variants, recombinant forms show discrete geographic
distribution patterns and contribute up to 20% of all global HIV infections.
Table 1.1: Worldwide Distribution of HIV (in Thousands).
Region People Living with HIV New HIV infections 2013 AIDS
2013 related
Total Children Total Children deaths 2013
(Total)
Sub- 24,700 2,900 (2,600 1,500 210 (180– 1,100
Saharan (23,500 – – 3,200) (1,300- 250) (1,000-
Africa 26,100) 1,600) 1,300)

Asia and the 4,800 (4,100 210 (190– 350 (250– 22 (18– 32) 250 (210–
Pacific -5,500) 270) 510) 290)

Latin 1,600 (1,400 35 (27– 54) 94 (71– 1.8 (<1 – 47 (39- 75)
America – 2,100) 170) 7.4)

Western and 2,300 (2,000 2.8 (2.3- 88 (44– <0.5 (<0.2- 27 (23– 34)
Central – 3,000) 3.6) 160) <0.5)
Europe and
N.America

Eastern 1100 (980– 14 (13– 14) 110 (86– 1 (<1– 1.2) 53 (43– 69)
Europe and 1300) 130)
Central Asia

Carribean 250 (230– 17 (14– 20) 12 (9.4 – <1 (<0.5 - 11 (8.3– 14)
280) 14) <1)

Middle East 230 (160– 16 (11– 22) 25 (14- 41) 2.3 (1.5- 15 (10– 21)
and North 330) 3.4)
Africa

Global 35,000 3,200 2,100 240 (210, – 1,500


(33,200 - (2,900- (1,900 - 280) (1,400 –
37,200) 3,500) 2,400) 1,700)

Source: UNAIDS 2013 Global Fact Sheet (UNAIDS, 2014)

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Phylogenetics employs molecular techniques to study evolutionary relationships among
organisms or genes by a combination of molecular biology and statistical techniques (Li,
1997), and has been useful in distinguishing genetically related groups of organisms.
Phylogenetic analysis of HIV strains reveals distinct clusters of HIV-1, with well-
defined patterns and trends of global distribution. Using these methods for example, the
global HIV-1 epidemic can be traced to the Democratic Republic of Congo (DRC).
Studies by Rambaut and colleagues showed that the DRC strains appeared to be basal
hence the hypotheses that each global subtype is a result of chance exportations of the
strains from founder viruses from the Democratic Republic of Congo into susceptible
risk groups followed by diversification (A Rambaut, Robertson, Pybus, Peeters, &
Holmes, 2001). These founder effects are what have given rise to the long evolutionary
branches observed for each of the subtypes (Worobey, 2007).
Circulating Recombinant Forms (CRFs) on the other hand are represented by mosaic
genomes that arose from different parent genomes (Robertson, Sharp, McCutchan, &
Hahn, 1995), hence unlike pure viral strains, their phylogenetic relationships are
dependent on the section of the genome being examined. Unique recombinant forms are
produced when two or more strains undergo recombination within an individual host,
before the resulting virus spreads beyond that host (Hoelsher & et al., 2001).
Overall, HIV genetic variations affect anti-retroviral drug response, viral transmission
and diagnostic and preventive vaccine strategies. Genetic variability is also important in
projecting morbidity and mortality and other key outcomes of infection (Hemelaar,
Gouws, Ghys, & Osmanov, 2011). For effective prevention, a HIV vaccine will have to
be developed that offers protection against at least majority of the circulating virus
strains (Thomson, Perez-Alvarez, & Najera, 2002).

1.2 Statement of the Problem


HIV-1 has a high mutation rate that is associated with continuous generation of
divergent virus populations. These mutants may have different rates of transmission and
virulence (Hemelaar et al., 2011; N. Kiwanuka et al., 2010).

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Like the global epidemic, the Kenyan HIV-1 epidemic follows a discrete but differential
genetic distribution pattern across regions of the country. Data shows that the variability
of the epidemic continues to evolve at the genetic level in Kenya, with new distribution
patterns of both pure and recombinant strains as well as declining dominance of HIV
subtype A (HIV-1A). This diversity affects intervention measures. For example, a
vaccine developed to prevent HIV-1 subtype A is unlikely to be effective against HIV-1
subtype C (Corey & McElrath, 2010). It is important to understand the present and
evolving trends of HIV-1 genotypes if we are to provide reliable data for disease
prognosis, treatment and prevention (Baeten et al., 2007; Vasan et al., 2006). This study
was conducted as part of an ongoing need to maintain a repository of knowledge on
virus heterogeneity and generate data that will inform policy, scientific and medical
strategies focused on controlling the spread and burden of HIV-1 and AIDS in Kenya.

1.3 Justification of the Study


An understanding of the molecular distribution of HIV-1 within Kenya is an important
public health concern. Since HIV-1 subtypes vary widely between geographical regions
and to some extent within localized populations, intervention strategies must be tailored
to unique epidemic characteristics of those regions. The counties were chosen from
facilities from the west of the country to the East. HIV-1 molecular epidemiological
studies have not been carried out in most of the health facilities under study (Kitengela
Health Centre, Naivasha District Hospital, Ndhiwa District Hospital, Ahero district
hospital)
In order to develop and implement successful intervention strategies, it is important that
the molecular trends and patterns of the HIV strains be constantly monitored and
updated. In addition, knowledge of HIV-1 diversity is pertinent to successful anti-
retroviral management and monitoring. HIV genotypic variability also affects viral
tropism, which has been shown to affect disease progression and pathogenicity (Pollakis
et al., 2001). In spite of the great significance of viral tropism, there is paucity of data on
HIV tropism in Kenya. Generating such data will significantly contribute to the
understanding of disease course and burden among the population, as well as inform

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alternative intervention strategies. This study avails new sequence information that
describes HIV diversity at genetic and protein level, which sequences have also been
deposited to the global databases to broaden scientific community‟s knowledge of local
HIV-1 epidemic.

1.4 Research Questions


1. What is the current genotype distribution pattern of HIV-1 in the Kenyan Counties of
Kisumu, Homabay, Kajiado, Nakuru, Kiambu, and Kilifi.
2. How does the genetic variability in HIV from these counties relate to HIV coreceptor
usage and viral envelope protein characteristics?
3. How does this genetic variability relate with disease outcomes and phenotypic
characteristics?

1.5 Objectives

1.5.1 General Objective


To describe the genetic diversity of HIV-1 within Kisumu, Homabay, Kajiado, Nakuru,
Kiambu, and Kilifi counties of Kenya.

1.5.2 Specific Objectives


1. To describe the genotypic distribution pattern of circulating HIV-1 strains from
Kisumu, Homabay, Kajiado, Nakuru, Kiambu, and Kilifi.
2. To determine the patterns of HIV-1 co-receptor usage of isolates from the study
counties.
3. To describe HIV-1 envelope diversity based on the patterns of Potential N-linked
glycosylation sites of different viral genotypes.

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CHAPTER TWO

LITERATURE REVIEW

2.1 The HIV-1 Structure and Genome Organization


HIV-1 and HIV-2 belong to the genus Lentivirus, family of Retroviridae and subfamily
Orthoretrovirinae (International Agency for Research on Cancer., 1996). Retroviruses
transcribe their genome from RNA into DNA using the enzyme reverse transcriptase.
The integrase enzyme then integrates the viral DNA into the host‟s genome, becoming
part of the host‟s cellular DNA. The integrated virus genome subsequently undergoes
replication during the natural cellular processes (Nisole & Saib, 2004).
Figure 2.1 shows a representation of a mature HIV virion. All lentiviruses are enveloped
by a lipid bilayer, which is derived from the membrane of the host cell. The surface
glycoproteins gp120 (labeled SU in the diagram; SU= surface glycoprotein or = sub-
unit) are anchored to the virus via the transmembrane (TM) glycoprotein gp41. A matrix
shell of the matrix-associated protein p17 (MA in Figure 2.1) lines the inner surface of
the viral membrane. At the viral core is the capsid protein p24 (CA), located at the
center of the virus. The capsid particle encapsulates two copies of the RNA viral
genome. The viral genome is stabilized within the capsid particle by the nucleocapsid
(NC) protein p7. The capsid particle also contains three essential virally encoded
enzymes: protease (PR), reverse transcriptase (RT) and integrase (IN). Virus particles
also package the accessory proteins, Nef, Vif and Vpr and three additional accessory
proteins Rev, Tat and Vpu (Turner & Summers, 1999).

6
Figure 2.1: Diagram of mature HIV virion (Steckbeck, Kuhlmann, & Montelaro,
2013)

The HIV genome consists of genes – env, gag and pol that code for structural proteins
(Figure 2.2) . The genome also has six regulatory genes - tat, rev, nef, vif, vpr, and vpu-
that code for proteins that control the ability of HIV to infect a cell, produce new copies
of virus, or cause disease (Bradac, Charles, Kristina, Catherine, & James, 2002).

Figure 2.2: The HIV-1 genome map based on HXB2 reference sequence. HIV
Immunology 2002 (Bradac et al., 2002).

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2.2 HIV-1 Diversity
The Human Immunodeficiency Virus is an RNA virus of the retrovirus family, with two
RNA strands making up its genome. Its genetic diversity is believed to derive from three
different sources; firstly, through multiple introductions of HIV into the human
population, secondly through the low fidelity and high recombinogenic power of the
viral reverse transcriptase enzyme and finally because of the high virus turnover (Castro-
Nallar, Keith A Crandall, & Pérez-Losada, 2012). This, together with high positive
selection pressure either due to the drugs or the host immune system results in
significant viral genetic variability (Ariën et al., 2005).
HIV-1 mutation rates average approximately 0.1-0.3 mutations per genome per
replication cycle (Abram, Ferris, Shao, Alvord, & Hughes, 2010), and is as a result of
high replication errors associated with the reverse transcriptase (RT) enzyme. During
reverse transcription, the enzyme undergoes a highly inefficient and error-prone
template switching between its two RNA genomic strands (Skar, Charlotte, & Jan,
2011). Genomic recombinations occur during this process in the order of about 0.14
recombinations per genome per replication cycle (Skar et al., 2011). These molecular
events produce quasi-viral species that proceed to superinfect the same cell or adjacent
host cells during host cell replication. Both recombination and RT-dependent replicative
error contribute enormously to HIV-1 diversity (Castro-Nallar et al., 2012). Although
selection forces in the host act to eliminate some of these viral mutants,
purifying/negative selection accompany these selections, generating persistent mutants
that are more potent and continue to propagate the epidemic at the population level (Skar
et al., 2011).
The evolutionary rate of HIV-1 has been shown to depend on the rate and mode of
spread (I. B. Maljkovic et al., 2007). Anti-retroviral treatment further exacerbates
evolution of HIV diversity through the selection of drug resistant mutants, particularly
individuals who adhere less to treatment or in settings where treatment is not optimal (B.
I. Maljkovic et al., 2009). In Kenya, HIV-1 infected people have continued to gain
increasing access to Antiretroviral (ARV) treatment (ART). This unprecedented access

8
to treatment poses significant challenges with treatment adherence as has recently been
reported among long-term ART patients (Ochieng et al., 2015). Under sustained ART,
more HIV-1 variants emerge either due to non-adherence or selective pressure that
produces drug resistant mutants (R.W. Lihana et al., 2011; Lwembe et al., 2007). These
„new‟ strains emerge from treated individuals and are propagated and transmitted across
population as new or sub-epidemics making future treatment of newly infected persons
with the same drugs impossible. Other studies have suggested that Highly Active
Antiretroviral Therapy (HAART) reduces the rate of evolution of HIV-1 Subtype B and
probably other subtypes, largely because therapy can significantly inhibit viral
replication and diminish accumulation of mutants (B. I. Maljkovic et al., 2009). As
treatment becomes more available in Kenya, the potential for viral evolution and
diversity become even more real.

2.3 Epidemiology of HIV strains


The Human Immunodeficiency Virus can be classified into two groups: HIV-1 and HIV-
2. It has become clear that HIV resulted from zoonotic transfers of viruses infecting
primates in Africa (Hahn, Shaw, De Cock, & Sharp, 2000). HIV-2 is mainly restricted to
Western Africa, and is not associated with AIDS epidemic (Santiago et al., 2005). The
HIV-1 group M pandemic is believed to have originated in the Congo River Basin and
has been traced by evolutionary analyses to having originated from there between the
years 1915- 1941 (Korber et al., 2000). HIV-1 is responsible for the global AIDS
epidemic and exists in diverse genetic forms distributed distinctly across the globe
(Hemelaar et al., 2011).
Group M of HIV-1 contributes to about 90% of HIV infections in the world (Spira,
Wainberg, Loemba, Turner, & Brenner, 2003). Hemelaar et al. carried out a global HIV-
1 epidemiology study on samples obtained between 2004 and 2007. Subtype B viruses
were found to be predominant in North America, the Caribbean, Latin America, western
and Central Europe and Australia. The epidemic in Eastern Europe and central Asia is
dominated by a mix of subtypes A and B. CRF01_AE predominates south and south-
east Asia regions while CRF07_BC, CRF08_BC, CRF01_AE and subtype B dominate

9
East Asia. The Middle East and North Africa are dominated by subtype B and CRFs
while West Africa features a highly heterogeneous epidemic with all the subtypes
represented but predominated by CRF02_AG and subtype G. East Africa on the other
hand has predominantly subtype A in circulation, with subtypes D, C and URFs also
emerging or increasing. Ethiopia, Southern Africa and India are predominated by
subtype C (Hemelaar et al., 2011). Overall, HIV-1 subtype B accounts for about 11% of
the global HIV burden, subtype C accounts for 48%, subtype A 12%, CRF02_AG
(8%), CRF01_AE (5%), subtype G (5%) and D (2%). Subtypes F, H, J and K together
cause less than 1% of infections worldwide. Other CRFs and URFs are each responsible
for 4% of global infections, bringing the combined total of worldwide CRFs to 16% and
all recombinants (CRFs along with URFs) to 20% (Hemelaar et al., 2011).
The explanation for the current global HIV-1 spread and diversity are multi-factorial:
population growth, founder effects, urbanization, migration and improved transport links
all have played major part (Hemelaar et al., 2011). It is thought that subtype C was
introduced into Southern Africa, India, China and Brazil through a preferential
introduction into a high risk heterosexual group (Walker, Pybus, Rambaut, & Holmes,
2005). In a study investigating the historical and evolutionary trends of HIV-1 in East
Africa, it was found that HIV-1A and D were introduced into the region after 1950 and
spread exponentially during the 1970s (Gray et al., 2009). The rapid growth and
divergence of the epidemic can be attributed to the strong interconnectivity between
population centers across the East African region.

The global distribution of HIV-1 is shown in Figure 2.3.

10
Figure 2.3: Regional distribution of HIV 1 subtypes and recombinants in 2000-2003
(a) and 2004-2007 (b) (Hemelaar et al., 2011).

11
2.4 The global HIV-1 genomic database and phylogenetic profiles
The HIV databases contain data on HIV genetic sequences, immunological epitopes,
vaccine trials and drug resistance-associated mutations. These platforms are useful when
analyzing HIV viral sequences for purposes of identification of specific viral genetic and
protein properties as well as deciphering epidemiologic trends. Various databases used
in HIV subtyping include the Los Alamos HIV database (lanl)
{http://www.hiv.lanl.gov/content/index}, The Stanford Drug Resistance Database
(http://hivdb.stanford.edu/), Geno2pheno (http://www.geno2pheno.org), Rega Subtyping
tool (http://www.bioafrica.net/rega-genotype/html/subtypinghiv.html) amongst others.
The criteria used for the current subtype classification is that subtypes are approximately
equidistant from one another in the envelope gene (env). Two or more samples are
required to define a new sequence subtype (Woutter, Buve, & Nkengasong, 1997).
Evolutionary analysis of HIV is then performed on the sequences by phylogenetic
inference, which captures the structure and genetic diversity of lineages using
phylogenetic trees. While the phylogenies depict major groups in data format, the tree
topologies show the evolutionary history of sequences and how these groups are related
in time. This far, phylogenetic inference has become useful in categorizing the major
groups and subtypes of HIV and their relationships (A. Rambaut, Posada, Crandall, &
Holmes, 2004).
Figure 2.4, adapted from (Archer, 2008) shows in (A) neighbor joining phylogenetic tree
reconstructed using global group M gp160 sequences sampled from LANL HIV
Sequence Database with the lettering representing the subtypes and * representing
bootstrap values greater than 90%. (B) shows the relationship between subtypes
diversity ratio and the quality of clustering as shown in (A Rambaut et al., 2001). The
group M phylogenetic tree can be described as having a double starburst appearance.

12
Figure 2.4: Neighbour joining phylogenetic tree for HIV-1 group M viruses
(Archer, 2008)

2.5 Kenyan HIV-1 Genetic Profile


According to the National AIDS Control Council (NACC) report 2014, the national
prevalence of HIV in Kenya is 6.04%, with the prevalence in women (7.6%) being
higher than that in men (5.6%). The epidemic is geographically diverse with prevalence
ranging from 25.7% in Homabay to 0.2% in Wajir (NACC., 2014). The predominant
subtype in Kenya is HIV-1A, although there is evidence of increasing prevalence of
subtypes C and D especially in regions around the northern border with Ethiopia (S. A.
Khamadi et al., 2005) and lately in the central regions (Kageha et al., 2012). There
have also been reports of a rise in inter-subtype recombinants in parts of Kenya
(Nyagaka et al., 2012; Songok et al., 2004). Subtype C has been found to be less fit
(hence lower disease progression) than all the other subtypes of Group M but has higher

13
transmissibility (Abraha et al., 2009; Asamoah-Odei, Garcia Calleja, & Boerma, 2004;
Gordon et al., 2003; Koch et al., 2001). The high transmission rate could be due to the
fact that subtype C viruses are predominantly R5-tropic; R5-tropic viruses having
greater transmissibility than the X4- tropic viruses (Abraha et al., 2009). Patients
infected with subtype C seem to have a high viral load but their CD4 counts reduce at a
lower rate than those of Subtype A and D therefore explaining their slower disease
progression. Slower disease progression and high viral load also means greater
opportunity for transmission (Abraha et al., 2009). On the other hand subtype D is
associated with fast disease progression and lower infectivity rates (Kaleebu et al., 2007;
N. Kiwanuka et al., 2010). One study evaluated the importance of recombinant forms
and showed that A/D recombinants are effectively transmitted from mother to child
(Steain, Wang, & Saksena, 2006).
Table 2.1 gives a summary of results from different studies within Kenya that give
subtype distribution in various regions. In practically all regions, subtype A is the major
subtype. One also observes regional differences in subtype distribution.

14
Table 2.1: Subtype Distribution in Kenya
Region in Kenya Common Subtype (in %)
A C D Others Recombinants
Northern (S. A. Khamadi et al., 57 27 9 7§
2005)
Coastal (S. A. Khamadi et al., 86 5 8 1*
2009)
Nairobi(R.W Lihana et al., 65 2 2 2* CRF02_AG - 7
2009c) A1/C - 9;
A1/D - 7;
C/D - 2;
A1/A2 - 2;
A1/D - 2
North Rift Valley 70 7 11 3- A2 A1/D - 4
(Nyagaka et al., 2012) 1* A1/C - 3
A2/C - 1
Western Kenya 51.4 1.4 9.4 4.1* All recombinants
(Micah et al., 2011) – 33.7
Kericho 56 5 10 All recombinants
(Arroyo et al., 2009) - 29
Kilifi 59 8 10 <1* All recombinants
(Hue S, 2011) -22
Kisumu 67.4 6.1 20.4 1.7*; 2.4§ Gag/Env:
(Yang et al., 2004) A2- D/A-8.7;
2.0 A/D-5.9,
C/A-2.4;
A/C-1.7
Nairobi 50 35.9 10.25 A1/D -3
(Kebira, Waihenya, & Khamadi,
2009)
Southern Kenya 56.9 2.4 2.4 A/D-14.6;
(Dowling et al., 2002) A/C-7.3;
A/G-2.4;
A/G- 2.4
C/D-2.4;
A/C/D-2.4
Nairobi 56.5 10.1 18.8 2.9* AD/CRF01_AE-
(Khoja et al., 2008) 4.34;
AC- 1.45;
AD -1.45
Central Kenya 69.8 11.5 18.7
(Kageha et al., 2012)
Notes: §, Unclassified; * Subtype G

15
2.6 Co-receptor Usage

2.6.1. HIV Phenotypes and Coreceptor Usage


Entry of the HIV virus into the host cell requires cooperate binding and fusion of the
viral surface envelope (env) glycoprotein trimer consisting of gp120 and gp41
heterodimers to host cell CD4 receptor. Binding to the CD4 receptor induces a
conformational change in Env that enables the engagement of a specific seven-
transmembrane chemokine receptor family on the cell surface (Craig B. Wilen, Tilton,
& Doms, 2012). HIV-1 specifically uses the CC β-chemokine receptor CCR5, and the
CXC α-chemokine receptor CXCR4. These are the predominant means of viral entry
into target CD4 T-cells (Pollakis et al., 2001). Based on co-receptor usage, HIV can be
characterized as CCR5 (or R5) tropic or CXCR4 (or X4) tropic strains. HIV-1 can
further be characterized phenotypically into synctium inducing (SI) and non synctium
inducing (NSI) viruses according to differences in cellular tropisms (Koot et al., 1992).
NSI viruses are majorly macrophage tropic and are normally observed in the early stages
of infection while SI viruses are T-Cell tropic and arise during late stages of disease
progression or infection (Archer, 2008; Berger et al., 1998). X4-tropic viruses are
largely of SI phenotype while R5-viruses are of NSI phenotype. Thus, co-receptor usage
has important association and link to both infection and disease outcome.

2.6.2 Mechanism of viral entry into host cell and coreceptor switching
The HIV-1 env gene can be divided into alternating constant (C) regions (C1 - C5) and
variable (V) regions (V1 -V5) (Starcich et al., 1986). The env region is important
because it contains an important target for immune response, determines coreceptor
specificity and shows variability that is highly phylogenetically informative
(Shankarappa et al., 1999). Crystal structure of gp120 shows that the constant and
variable regions can be organized into 4 structural domains i.e. the inner and outer
domains, a 4-stranded bridging sheet and the V1/V2. On binding to the CD4 receptor,
the inner domain of gp120 undergoes major structural changes that allow for formation
of the bridging sheet while the majority of the outer domain appears to remain

16
unchanged. Subsequent co-receptor binding at areas on the bridging sheet to the outer
domain of the virus V3 loop causes additional gp120 conformational changes that result
in the ultimate dissociation of gp120 from gp41 and transition of gp41 into a structural
form that allow for viral-host membrane fusion (Wang et al., 2013). The virus V1V2
region has been shown to provide the genotypic pattern for R5X4 dual tropism, although
it does not affect CCR5 utilization (Pollakis et al., 2001). The overall amino acid charge
of the V3 region has an effect on the biological phenotype with a higher positive charge
favouring the SI phenotype and a higher utilization of CXCR4 coreceptor. The loss of an
N-linked glycosite in the V3 region together with the higher positive charge has been
shown to result in strong CXCR4 tropism and is thought to contribute to virus switching
from R5 to the X4 phenotype (Pollakis et al., 2001).
The co-receptors are useful target for antiretroviral drugs and candidate therapeutics
(Kalinina, Pfeifer, & Lengauer, 2013). So far only CCR5 coreceptor blockers (entry
inhibitors) have been developed, for example Maraviroc, which act by blocking the
CCR5 coreceptor hence inhibiting entry. Although some candidate compounds have
been shown to block viral entry via CXCR4 co-receptor, these have not been developed
into antiretroviral agents because CXCR4 is critical for haematopoesis, cardiac function
and cerebellar development hence using it as a target can lead to serious side effects
(Archer, 2008). Coreceptor usage can be measured or deduced by using either
phenotypic assays or genotypic assays (Mulinge et al., 2013). This study used the
genotypic method, relying on the Geno2Pheno platform which integrates position matrix
analyses for CCR5 and CXCR4 at specific amino acids and glycosylation sites (Sing,
Beerenwinkel, & Lengauer, 2004).

2.7 HIV Envelop Diversity and Potential N-Linked Glycosylation Patterns


The HIV-1 gp120 is heavily glycosylated by the infected host, with the glycan moieties
accounting for up to 50% of its total mass (Wang et al., 2013). N-Linked glycosylation
sites are also referred to as sequons. The position and number of N-Linked
oligosaccharides attached to a protein have a profound effect on the protein structure,
expression and function (Kasturi, Chen, & Shakin-Eshleman, 1997). An amino acid

17
sequence with pattern N-X-S or N-X-T is required for glycosylation to take place. N in
this context refers to Asparagine, X refers to any amino acid except proline, S refers to
Serine and T refers to Threonine. A sequon will not be glycosylated if it contains Proline
(Ming, Gaschen, & et al., 2004). Sequons are important because they have an effect on
immunogenicity. A change of sequons in the HIV envelope protein gp41 for example
can induce a conformational change in the associated HIV gp120 that will dramatically
diminish the binding of specific gp120 antibodies (Si, Cayabyab, & Sodroski, 2001).
These patterns also influence receptor binding and the phenotypic properties of the virus
(Pollakis et al., 2001). They could also cause T cell immune response escape (Botarelli
et al., 1991; Ferris et al., 1999). Glycosylation of gp120 can change exposure of
neutralizing antibody epitopes. A glycosylation site can therefore provide regional
protection from antibodies the extent of which depends on the various viral subtypes.
For example, the absence of a sequon near the base of the V3 loop in CRF01 strains of
HIV has been correlated with rapid amino acid substitution and positive selection
(Kalish et al., 2002). Glycosylation at position 301 at the stem of the V3 loop is thought
to play a role in stabilizing the V3 and in modulating coreceptor binding. Mutation at
this point results in a drastic decrease in viral infectivity probably due to reduced
coreceptor binding (Wang et al., 2013). A study by Chohan and colleagues indicated
that there is an apparent selection for HIV subtype A and C variants that are less
glycosylated and with shorter V1-V2 loop sequences (Chohan et al., 2005). It has also
been found that potential N-linked glycosylation sites (PNGs) interact among
themselves with negative or mutually exclusive forces occurring between sites of the
same location and positive interaction occurring between sites separated by greater
distances (Poon, Lewis, Kosakovsky, & Frost, 2007). The number of N-linked
glycosylation sites have also been seen to be significantly different between the plasma
and cellular compartments with the plasma compartment showing higher numbers of
PNGs (Ho et al., 2008). Thus, N-linked glycosylation of the HIV-1 envelope
glycoprotein is one of the most protective mechanisms to overcome in order to develop
an effective neutralizing antibody inducing vaccine (Poon et al., 2007). This study also

18
aimed to map out the PNGs of various HIV-1 isolates and compare these patterns
between cellular and acellular blood compartments.

19
CHAPTER THREE

MATERIALS AND METHODS

3.1 Study design


This was a cross-sectional study in which fresh blood samples and demographic
information were collected from subjects at designated HIV comprehensive care centers
around the country.

3.2 Study site


Subjects were recruited from HIV Comprehensive Care Centers in the counties of
Kisumu (Ahero District Hospital), Homabay (Ndhiwa District Hospital) Nakuru
(Naivasha District Hospital), Kajiado (Kitengela Health Centre), Kilifi (Malindi District
Hospital) and Kiambu (Kiambu District Hospital). The sites were chosen to cover areas
with the highest prevalence of HIV (Homabay and Kisumu) located in the Western
region to regions with lower prevalence (Kiambu, Nakuru and Kajiado). Kilifi was
chosen because the diversity of subtypes in this region and due to the fact that it is on the
Eastern coast of Kenya. Kitengela Health Centre in Kajiado was also chosen because the
region where it is located is an important stop-over for long distance drivers. The
laboratory analysis was done at KEMRI-CVR, Strathmore CREATES (Centre for
Research in Therapeutic Sciences) laboratory and at the African Centre for Clinical
Trials. According to the National Aids Control Council 2014 report, the HIV prevalence
in Kisumu is 19.3%, Homabay 25.7%, Nakuru 5.3%, Kajiado 4.4%, Kiambu 3.8% and
Kilifi 4.4%. (NACC., 2014). Figure 3.1 gives the Kenya County HIV-1 prevalence.

20
study locations

Figure 3.1: Map of Kenya Counties with corresponding HIV prevalence


Source: (NACC., 2014)

21
3.3 Study population
The study was on HIV-1 positive patients (see sample size determination) aged 13-64
years who were receiving antiretroviral treatment (ART) and care at comprehensive care
centres (CCC) of the respective counties.. To get a more homogenous group, the
paediatrics (12 years and below) and geriatrics (65 years and above) were excluded. The
patients were consented and enrolled between April and August 2013.

3.3.1 Inclusion Criteria


Consenting individuals who:
1. Were receiving or not receiving anti-retroviral drugs and
2. Were aged 13-64 years and appearing at the participating facilities for care and
3. Had a determined HIV status and enrolled for care within the HIV-care facilities.

3.3.2 Exclusion criteria


1. All non-consenting patients
2. Patients whose place of residence was not within the respective counties.

3.4 Sampling

3.4.1 Sample Size Determination


This was a cross-sectional survey and therefore the following Cochran‟s formula was
used.
n = zα2*p*q
d2
zα= z score for α of 0.05
p= national prevalence of HIV = 5.6% from KAIS-2012 (NASCOP., 2014)
q= 1-p
d= precision (5% precision will be used)
n=1.962*0.056*0.944 = 0.2031 = 81.23
0.052 0.0025
Minimum sample size needed for statistical representativeness of population was 81.

22
3.4.2 Sampling Procedure
Upon consent, a questionnaire was administered to all subjects to collect demographic
and relevant clinical information including age, sex, sex partnership, occupation,
antiretroviral drug use profile, ART regimen, and travel history. Patient files were also
examined to collect information on CD4 counts and viral load where available.

3.5 Ethical Considerations


This study was conducted in accordance with the Helsinki Declaration of 1975, (revised
in 2000) (World Medical Association, 2000) and after approval by the Scientific
Steering Committee (SSC) and the Ethical Review Committee (ERC) of the Kenya
Medical Research Institute (ERC/SSC protocol #2477). Participants were informed
adequately about the study and their written consent obtained voluntarily before
recruitment. Patient data was coded to ensure confidentiality

3.6 Laboratory and Analytical Procedures

3.6.1 Sample processing


Sample processing was done according to KEMRI CVR Standard Operating Procedures
(SOPs). Five milliliters of venous blood was drawn into EDTA vacutainer tubes from
each patient. Plasma was obtained by centrifuging the blood at 3000 revolutions per
minute (rpm) or 900 x g for 10 minutes. Aliquots of the plasma were frozen at -80oC
until needed for RNA processing. The cellular portion of the blood was lysed to obtain
Peripheral Blood Mononuclear Cells (PBMCs). The lysis was done by adding 12 ml of
0.84% ammonium chloride to the plasma-free blood in a 15 ml falcon tube, vortexing
and incubating for 10 minutes at 37oC. The tubes were then centrifuged at 900 x g for 10
min and the supernatant discarded. This was repeated 3 times. The cell pellets were
washed by re-suspending in 1 ml of Phosphate Buffer Saline (PBS, pH 7.4) and
centrifuging at 900 x g for 5 minutes in 1.5 ml eppendorf tubes. The resulting PBMCs
were frozen as pellets at -80oC until needed for DNA processing.

23
3.6.2 Viral Load Determination
Only patient samples with viral load above 1000 RNA copies/ml were considered for
RNA extraction and reverse transcriptase polymerase chain reaction (RT-PCR). Viral
load was determined using m2000 Abbott RealTime HIV-1 assay following
manufacturer‟s protocol (Abbott Laboratories, Abbott Park, Illinois). Briefly, internal
control RNA was added to 200µl of plasma and loaded onto m2000sp instrument for
RNA extraction. The range for the limits of detection was between 40 and 10,000,000
HIV-1 RNA copies. Undetectable VL was reported as 39 copies. All VL assays were
conducted at the Early Infant Diagnosis laboratory of the Kenya Medical Research
Institute (KEMRI).

3.6.3 Nucleic Acid Preparation


DNA was extracted from samples that had between undetectable viral load and up to a
maximum of 1000 HIV-1 RNA copies/ml blood.
QIAamp DNA kit (Qiagen, Maryland USA) was used for DNA extraction and QIAamp
RNA (Qiagen, Maryland USA) kit was used for RNA extraction. Before starting nucleic
acid extraction, all samples and distilled water were equilibrated to room temperature.
The oven was heated to 56°C and maintained at this temperature. Buffer AW1, Buffer
AW2 were prepared by adding the indicated amount of absolute ethanol. All
centrifugation steps were carried out at room temperature (15 – 25°C).

3.6.3.1 DNA Extraction Procedure


DNA extraction was done using Qiamp DNA Kit following instructions in the
QIAamp® DNA Mini and Blood Mini Handbook. (Qiagen®, 2010). Briefly, Qiagen
Protease (Qiagen, Maryland, USA) was prepared by adding the required amount of
protease solvent to the tube containing the enzyme.

Twenty microlitres of QIAGEN Protease (or proteinase K) was pipetted into the bottom
of a 1.5 ml microcentrifuge tube and 200 μl sample (PBMCs mixed with PBS pH 7.4)
was added to the microcentrifuge tube. Two hundred microlitres of buffer AL was then

24
added to the sample and mixed by pulse-vortexing for 15 s. The mixture was then
incubated at 56°C for 10 min. The 1.5 ml microcentrifuge tube was briefly centrifuged
to remove drops from the inside of the lid.

Two hundred microlitres of ethanol (96 – 100%) was added to the sample and mixed
again by pulse-vortexing for 15 s. After mixing, the 1.5 ml microcentrifuge tube was
briefly centrifuged to remove drops from the inside of the lid. The mixture was then
applied to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the
rim. The cap was closed and the mixture centrifuged at 6000 x g for 1 min.

The QIAamp Mini spin column was placed in a clean 2 ml collection tube, and the tube
containing the filtrate was discarded. The QIAamp Mini spin column was carefully
opened and 500 μl Buffer AW1 was added without wetting the rim. The cap was closed
and the column centrifuged at 6000 x g for 1 min.

The QIAamp Mini spin column was then placed in a clean 2 ml collection tube and the
collection tube containing the filtrate discarded. The QIAamp Mini spin column was
carefully opened and 500 μl of buffer AW2 added without wetting the rim. The cap was
closed and the column centrifuged at full speed (20,000 x g) for 3 min.

The QIAamp Mini spin column was then placed in a new 2 ml collection tube and the
old collection tube with the filtrate was discarded. The column was centrifuged again at
full speed for 1 min. The QIAamp Mini spin column was then placed in a clean 1.5 ml
microcentrifuge tube and the collection tube containing the filtrate was discarded. The
QIAamp Mini spin column was carefully opened and 40 μl of distilled water (RNAse,
DNAse free) was carefully pipetted to the centre of the membrane without touching it.

The column was incubated at room temperature (15 – 25°C) for 2 min, and then
centrifuged at 6000 x g for 1 min. The column was discarded and the 1.5 ml

25
microcentrifuge tube contained extracted DNA was tightly closed. To prevent frequent
thawing that affects the integrity of the DNA, the DNA extracted was divided into two
portions and placed in two separate microcentrifuge tubes and stored at -80oC until
required.

3.6.3.2 RNA Extraction Procedure


Extraction of RNA from plasma samples was carried following manufacturer‟s
instructions using the QIAamp® Viral RNA Mini Handbook (Qiagen, 2012). Briefly,
310 microlitres of buffer AVE was added to the tube containing 310 μg lyophilized
carrier RNA to obtain a solution of 1 μg/μl. The carrier RNA was dissolved thoroughly
and divided into conveniently sized aliquots and stored at –20°C.

Carrier RNA was mixed with buffer AVL gently by inverting the tube 10 times,
following manufacturer‟s instructions. Vortexing was not done to avoid foaming.
Five hundred and sixty microlitres of prepared buffer AVL containing carrier RNA was
pipetted into a 1.5 ml microcentrifuge tube. One hundred and forty microlitres of plasma
was added to the buffer AVL–carrier RNA in the microcentrifuge tube and mixed by
pulse-vortexing for 15 s.

The mixture was then incubated at room temperature (15 – 25°C) for 10 min. The tube
was briefly centrifuged to remove drops from the inside of the lid. Five hundred and
sixty microlitres of ethanol (96 – 100%) was added to the sample, and mixed by pulse-
vortexing for 15 s. After mixing, the tube was briefly centrifuged to remove drops from
inside the lid.

Six hundred and thirty microlitres of the resulting lysate solution was carefully applied
to the QIAamp Mini column (in a 2 ml collection tube) without wetting the rim. The cap
was tightly closed to avoid cross-contamination during centrifugation. The column was
centrifuged at 6000 x g for 1 min. The QIAamp Mini column was then placed into a

26
clean 2 ml collection tube and the tube containing the filtrate discarded. This step was
repeated with the remaining lysate.

The QIAamp Mini column was carefully opened and 500 μl of Buffer AW1 added. The
cap was closed and the column centrifuged at 6000 x g for 1 min. The QIAamp Mini
column was placed in a clean 2 ml collection tube and the tube containing the filtrate
discarded.

The QIAamp Mini column was carefully opened and 500 μl of Buffer AW2 added. The
cap was closed and the column centrifuged at full speed (20,000 x g) for 3 min.
The QIAamp Mini spin column was then placed in a new 2 ml collection tube and the
old collection tube with the filtrate was discarded. The column was centrifuged again at
full speed for 1 min.

The QIAamp Mini spin column was again placed in a clean 1.5 ml microcentrifuge tube
and the collection tube containing the filtrate was discarded. The QIAamp Mini spin
column was carefully opened and 40 μl of distilled water (RNAse, DNAse free water)
was carefully pipetted to the centre of the membrane without touching it. The column
was incubated at room temperature (15 – 25°C) for 2 min, and then centrifuged at 6000
x g for 1 min. The column was discarded and the 1.5ml microcentrifuge tube was tightly
closed.

To prevent frequent thawing that affects the integrity of the RNA, the RNA extracted
was divided into three portions and placed in three separate microcentrifuge tubes and
stored at -80oC until use.

3.6.4 Choice of Primers for Polymerase Chain Reaction (PCR)


Gene specific primers were designed that targeted a 701 base pair (bp) region of reverse
transcriptase (RT) region and corresponding to nucleotides nt(nucleotide)2480- nt3180
of the HIV HXB2 pol gene reference sequence. For envelope gene, the primers targeted

27
a 546bp region of C2V3 gene segment corresponding to nt6975-nt7520 on the HIV
HXB2. The primer sequences are shown in Table 3.1.

Table 3.1: Pool of HIV specific primers used for amplification of env and pol RT
regions by polymerase chain reaction (PCR)
HIV-1 Primer Sequence Purpose Citation
region
Envelope M5_5‟- CCAATTCCCATACATTATTGTGCCCCAGCTGG -3‟ Round
(C2-V3, nt (F) 1PCR
6975– M10_5‟- CCAATTGTCCCTCATATCTCCTCCTCCAGG -3‟ (R (S.A
7520 ) Khamadi
HXB2); et al.,
546bp M3_5‟- GTCAGCACAGTACAATGCACACATGG-3‟ (F) Nested 2008)
M8_5‟- CCTTGGATGGGAGGGGCATACATTGC-3‟ (R) PCR/
sequencing

Pol-RT RT18_5‟-GGAAACCAAAAATGATAGGGGGAATTGGAGG - Round 1 (Lwembe


(nt 2480– 3‟ (F) PCR et al.,
3180 KS104 _5‟- TGACTTGCCCAATTTAGTTTTCCCACTAA-3‟ 2007)
HXB2); (R)
701bp
KS101_5‟- Nested
GTAGGACCTACACCTGTTCAACATAATTGGAAG-3‟ (F) PCR/
KS102_5‟- sequencing
CCCATCCAAAGAAATGGAGGAGGTTCTTTCTGATG -3‟
(R)
Key: (F)- Forward Primer; (R)- Reverse Primer

3.6.5 Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Round 1


PCR for RNA Samples
Plasma-derived RNA samples were reverse transcribed using Qiagen OneStep RT-PCR
kit (Qiagen, Maryland, USA).

28
Briefly, each RT-PCR reaction included 1x PCR buffer that contained 1.5 mM MgCl2,
0.4 mM dNTPs, 0.6 µM forward and reverse primers, 1 µl enzyme mix, 5 µl of RNA
template, 5 units RNAseOut and nuclease-free water in a final volume of 25 µl. Reverse
transcription was done at 50oC for 30 minutes followed immediately by PCR
amplification cycles comprising denaturation at 95 oC for 15 min and 38 PCR cycles of
denaturation at 94oC for 30 s; annealing for 45 s at 58oC for env primers or 55oC for pol-
RT primers and extension at 72oC for 1min. A final extension was done for 10 min at
72oC.

3.6.6 Nested PCR for RNA Samples


Nested PCR was carried out using HotStar Taq polymerase (New England Biolabs,
Massachusetts, USA). The nested PCR components included the Taq enzyme
(0.625units/25µl), 2 mM MgCl2, 0.2 mM dNTPs, 0.5 µM forward and reverse primers,
1x PCR buffer and nuclease-free water in a final reaction volume of 25 µl. Primers used
for the nested PCR were M3 and M8 for env and KS101 and KS102 for pol-RT genes
(Table 3.1). The PCR conditions were: Initial denaturation at 95oC for 5 min; 38 cycles
of denaturation at 95oC for 30 s, annealing at 56oC for 45 s for both env and pol RT
regions, and extension at 68oC for 45 s. Final extension was at 68oC for 10 min.

3.6.7 DNA PCR


Round 1 DNA PCR was carried out using HotStar Taq polymerase (New England
Biolabs, Massachusetts, USA). The PCR components included Taq enzyme
(0.625units/25µl), 2 mM MgCl2, 0.2 mM dNTPs, 0.5 µM forward and reverse primers
(M5 and M10 for envelope and RT18 and KS104 for pol-RT genes) 1x PCR buffer, 1-
3µl of the template and nuclease-free water in a final reaction volume of 25 µl. Initial
denaturation was done at 95oC for 5 min; 38 cycles of denaturation at 95 oC for 30 s,
annealing at 58oC for 45 s for env and 55oC for pol RT regions, and extension at 68oC for
45 s. Final extension was at 68oC for 10 min.

29
Round 2 PCR was also carried out using HotStar Taq polymerase (New England
Biolabs, Massachusetts, USA). The PCR components included the Taq enzyme
(0.625units/25µl), 2 mM MgCl2, 0.2 mM dNTPs, 0.5 µM forward and reverse primers
(M3 and M8 for envelope and KS101 and KS102 for pol-RT genes) 1x PCR buffer, 1-
3µl of the round 1 amplicon and nuclease-free water in a final reaction volume of 25 µl.
Initial denaturation was done at 95oC for 5 min; 38 cycles of denaturation at 95 oC for 30
s, annealing at 56oC for 45 s for both env and pol RT regions, and extension at 68oC for
45 s. Final extension was at 68oC for 10 min.

3.6.8 Gel Electrophoresis


A 1.6% agarose gel matrix was made by mixing 0.64 g of agarose powder (Invitrogen,
USA) with 40 ml of 1X Tris EDTA electrophoresis buffer. This was heated in a
microwave until the powder had dissolved and then cooled to about 50oC. Two µl of
ethidium bromide was then added to the mixture. Ethidium bromide intercalates into the
DNA and fluoresces under UV light and hence allows visualization of nucleic acid after
electrophoresis.

The solution was poured into a casting tray containing a sample comb and was
polymerized for 20 min at room temperature. The sample comb was removed once the
gel had polymerized and the gel was inserted into an electrophoresis chamber and
covered with buffer solution. The first well was reserved for the DNA ladder. A total
volume of 6 µl was used that comprised for the first well, 1 µl DNA ladder, 1 µl loading
dye and 4 µl of distilled DNAse free, RNAse free water. The samples were loaded on
the remaining wells by mixing 5 µl of the amplicon with 1 µl of loading dye. Since
DNA is negatively charged, the gel was placed in the gel tank with the wells facing the
cathode so that the current was run towards the anode (positive pole). A current of 100
volts was applied for 30 min.

The larger DNA fragments move more slowly through the agarose gel matrix than the
smaller fragments and in this way, the DNA fragments are fractionated, The DNA ladder

30
helps one to identify the size of the DNA fragments. The gel was visualized under UV
light.

3.6.9 Purification and Sequencing


Amplified products were purified using QIAquick PCR purification kit (Qiagen,
Maryland USA) so as to remove primers, nucleotides, enzymes and other impurities
from DNA samples that could interfere with sequencing. Briefly, binding buffer was
added directly to the PCR sample and the mixture applied to the QIAquick spin column.

The binding buffer contains a pH indicator that allows for easy determination of the
optimal pH for DNA binding. Nucleic acids were adsorbed to the silica membrane in the
high-salt conditions provided by the buffer. Impurities were washed away and pure
DNA was eluted with 40 µl of water. Two microlitres was examined for presence of
DNA and purity using a nanodrop spectrophotometer using Nanodrop2000 looking out
for A260/A280 ratios of between 1.7 and 1.9. Due to the fact that the Macrogen
sequencing facility in Amsterdam is efficient in giving high quality sequencing results in
a short time and at a reasonable price, purified samples were sent to macrogen.

Twenty microlitres was packed for shipping to the Netherlands and sequenced following
the macrogen standard sanger sequencing procedure using the ABI3730XL genetic
analyzer. The sequencing required of 10 µl of 20ng/µl PCR product and 10 µl of
5pmol/µl nested PCR primers.

3.6.10: HIV-1 Subtyping and Subtype Analysis


Each partial sequence was manually inspected, aligned and edited using Bioedit
platform (http://www.mbio.ncsu.edu/bioedit/ from North Carolina State University)
(Hall, 1999). Initial pairwise alignment was done to join the reverse and forward
sequences. The joined sequences were subsequently subjected to multiple sequence
alignment against standard reference sequences from the Los Alamos National
Laboratory HIV database using Clustal W (Larkin et al., 2007). At least one reference

31
sequence for each HIV-1 subtype and the HXB2 reference sequence were included with
the query sequences to generate the multiple sequence alignments. A consensus
sequence was generated of all the alignments and used together with HIV HXB2
reference sequence, to facilitate further editing. Aligned sequences were transferred to
MEGA 6 (Molecular Evolutionary Genetic Analysis 6) sequence analysis platform
(Koichiro, Glen, Daniel, Alan, & Sudhir, 2013) for phylogenetic analysis. Phylogenetic
trees were generated using the neighbor joining method, the test of phylogeny being the
bootstrap method using 1000 replications. The substitution model used was the Kimura
2-parameter method. The generated trees were used to infer and assign HIV-1 subtypes.

For purposes of concordance analysis of different subtyping tools, multiple additional


subtyping tools were used and compared with the phylogenetic method. These were:
NCBI (http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi), Stanford for
Pol RT (Rhee et al., 2003; Shafer, 2006); Geno2Pheno (for Pol RT) (Lengauer & Sing,
2006); COMET (Struck, Ternes, Schmit, & Bercoff, 2014); jpHMM – jumping profile
hidden markov model (Schultz et al., 2009); REGA (from Rega Institute) (Alcantara et
al., 2009; de Oliveira et al., 2005) and SQUEAL (Delport, Poon, Frost, & Pond, 2010).
It was also hypothesized that HIV-1 genotypes could vary genetically across the
different blood compartments to reflect the uniqueness of these environments.
Therefore, compartmentalization of HIV genetic variants was examined, using the
cellular and cell-free compartments as reference. Virus isolates from cellular (DNA) and
cell-free (plasma RNA) blood were sequenced on the pol-RT and env genes, and
distribution of genotypes compared between the two compartments. Finally, the
geographic distribution of subtypes by county was determined.

3.6.11: Determination of Coreceptor Usage


To determine co-receptor usage, edited sequences that cover the V3 region were input
into the relevant section of the geno2pheno phenotype prediction tool (Lengauer,
Sander, Sierra, Thielen, & Kaiser, 2007). Available genotypic tools were developed
primarily for detection of subtype B HIV viruses, which are common in Europe and the

32
U.S.A, and consequently are biased against non-clade B isolates (Garrido et al., 2008).
Geno2Pheno was used for determination of tropism in this study because it has
previously been found to be sensitive to non clade B X4 variants (Garrido et al., 2008).

To determine virus co-receptor usage or viral tropism, a false positive rate (FPR) cut-off
of 10% (FPR10) was first applied, being the standard recommendation of the „European
Consensus Group on clinical management of HIV-1 tropism testing‟. Since only a single
sequence for each virus isolate was generated, analysis was repeated with FPR set at
20% (FPR20) to increase fidelity for correctly assigning X4-phenotype, and at 5% FPR
(FPR5) based on recent findings from subtype C HIV-1 phenotyping that revealed an
increased confidence at identifying CXCR4-usage in both CXCR4-exclusive and dual
tropic variants (Crous, Krishna, & Travers, 2012). Variations in these strategies were
finally compared. In all cases, values below set cut-off were considered to be predictive
of an X4 virus.

3.6.12: Determination of envelope protein diversity


The number of potential N-Glycosylation sites (PNGs) was determined from env
sequences using the Los Alamos HIV database platform (Zhang et al., 2004). Briefly,
viral sequences generated were aligned and edited using BioEdit as described in section
3.4.7. The curated sequences were then applied to the Los Alamos database. The nucleic
acid sequences were first translated into amino acids sequence using the TRANSLATE
tool within the Los Alamos Database
(http://www.hiv.lanl.gov/content/sequence/TRANSLATE/translate.html). The generated
amino acid sequences were then aligned to HXB2 reference sequence using HIVAlign
tool (http://www.hiv.lanl.gov/content/sequence/VIRALIGN/viralign.html) and then
applied to the N-Glycosite tool (Zhang et al., 2004) to generate an array of PNG. The
number of PNGs was assigned based on the NXT, NXS and the contiguous (NNXT(S))
patterns. PNG site patterns were compared across different viral strains.

33
3.7 Data Management and Analysis
A computerised data entry system was used to enter and store all subject information
using unique subject identity codes. Initial entries were accomplished at the point of data
collection by two independent personnel to ensure accuracy and security. Practical
information on subjects‟ locality, unique identification number, occupation and age were
coded and double entered into electronic storage servers and computers. Analysis of data
was done using the statistical package for social sciences (SPSS).

34
CHAPTER FOUR

RESULTS

4.1 Patient Characteristics


In this study, 81 patients were recruited from six facilities around the country. From the
81 isolates, 19 amplified for both the polRT and C2V3 genes. Patients were on the
following treatment regimen: 1.2% (1/81) of the patients were on ABC+3TC+NVP,
33.3% (27/81) were on AZT+3TC+NVP/EFV, 19.8% (16/81) were on
D4T+3TC+NVP/EFV and 24.7% (20/81) were on TDF+3TC+NVP/EFV, with NVP and
EFV being used as alternate non-nucleoside reverse transcriptase inhibitors (NNRTI).
Eight of the 81 patients (9.9%) were not on ART and ART regimen for 11.1% (9/81) of
the patients was not recorded (NR). By gender, 32.1% (26/81) of the patients were
males, 64.2% (52/81) were females and 3.7% (3/81) were not recorded. The distribution
of the patients by age was 7.4% (6/81) below 25 years old, 33.3% (27/81) between 25
and 35 years, 44.5% (36/81) between 36 and 50 years, 11.1% (9/81) above 50 years and
3.7% (3/81) were not recorded. The treatment and demographic data are presented as
part of table 4.1 for pol RT sequences and table 4.2 for C2V3 sequences.

Overall, patients had been on treatment for a median length of 40 months (Range: 0 -
102 months). The CD4 count was obtained from patient records. CD4 T-cell count was
less than 350/mm3 for approximately 50% of the patients, with 27% having a CD4 count
greater than 500/mm3. About 60% of the patients had their viral load below detectable
levels (39 HIV-1 RNA copies/ml blood). Of the 100 starting patient samples, DNA was
extracted from PBMC from 48 patients while RNA was extracted from plasma from 33
patients for a total of 81 nucleic acid material. Fifty-nine of the 81 patient samples were
successfully amplified with envelope-specific primers (Table 4.2), 42 of these being
from PBMC derived DNA and 17 from plasma derived RNA. Another 41 of the 81

35
samples (Table 4.1) (19 derived from PBMC DNA and 22 from plasma derived RNA)
were successfully PCR-amplified on the pol-RT gene. Nineteen samples amplified for
both pol RT and C2V3 regions.

36
Table 4.1: Patient characteristics based on pol RT phylogenetic subtypes
Subtype (Number of Sequences)
Unassigned A1 A2 A2D C D H Total
Region
Homabay 1 1
Kisumu 2 1 3
Kajiado 7 1 1 9
Kiambu 4 1 5
Malindi 1 6 1 1 3 12
Naivasha 10 1 11
Total 1 30 1 1 2 5 1 41
Sex
Male 11 1 12
Female 1 19 1 1 2 3 1 28
Not recorded 1 1
Total 1 30 1 1 2 5 1 41
ART Regimen
ABC/3TC/NVP 1 1
AZT/3TC/EFV 1 1
AZT/3TC/NVP 7 2 9
D4T/3TC/NVP 1 6 1 8
None 4 1 5
NR 4 1 5
TDF/3TC/EFV 4 2 1 7
TDF/3TC/NVP 4 1 5
Total 1 30 1 1 2 5 1 41
Age
Below 25 2 1 3
25-35 11 1 1 1 4 18
36-50 1 14 1 16
Above 50 3 3
Not recorded 1 1
Total 1 30 1 1 2 5 1 41
This table shows the HIV subtypes from 41 patients recruited from Kisumu, Kiambu, Kitengela, Malindi,
Homa-Bay and Naivasha, derived from the pol-RT gene.

37
Table 4.2: Patient characteristics based on C2V3 phylogenetic subtypes
Subtype (No. of Sequences)
A1 A2 C D N.D Total
Region
Homabay 2 1 3
Kisumu 10 1 2 2 1 16
Kajiado 11 2 1 14
Kiambu 2 2 4
Malindi 6 1 7
Nakuru 10 2 1 2 15
Total 41 4 5 5 4 59
Sex
Female 25 1 3 4 3 36
Male 14 3 2 1 1 21
Not recorded 2 2
Total 41 4 5 5 4 59
ART Regimen
ABC/3TC/NVP
AZT/3TC/EFV 3 1 4
AZT/3TC/NVP 11 1 2 3 2 19
D4T/3TC/NVP 6 1 1 2 1 11
None 5 1 6
Not recorded 6 6
TDF/3TC/EFV 2 1 3
TDF/3TC/NVP 8 1 1 10
Total 41 4 5 5 4 59
Age
Below 25 5 5
25-35 15 1 1 1 2 20
36-50 14 2 4 3 1 24
Above 50 5 1 1 1 8
Not recorded 2 2
Total 41 4 5 5 4 59
This table shows HIV-1 genotypes based on envelope gene as obtained using Phylogeny for 59 patients
who were recruited from Kisumu, Kiambu, Malindi, Homabay and Naivasha and Kitengela. Key: N.D,
Not determined (these sequences were found to be too short by the publisher (AIDS Research and Human
Retroviruses) and were not included in the Phylogenetic Analysis).

38
4.2 HIV-1 subtyping

4.2.1 Phylogenetic Analysis

4.2.1.1. Phylogenetic analysis for the envelope C2V3 Sequences


From the 59 sequences, four were deemed to be too short by the publisher (AIDS
Research and Human Retroviruses) and were not included in the phylogenetic analysis.
Of the remaining 55 sequences (Figure 4.1), 41/55 (74.5%) of sequences clustered with
subtype A1 and therefore were determined to be subtype A1 by phylogeny. Four of 55
(7.3%) clustered with subtype A2, 9.1% (5/55) clustered with subtype C and 9.1% (5/55)
clustered with subtype D reference sequences, and were assigned corresponding
subtypes.

39
Figure 4.1: Phylogenetic Relationships of HIV-1 envelope C2V3 gene
The isolates
evolutionary distances of the various virus isolates40
were inferred using the Neighbour-Joining method. The
evolutionary distances were computed using the Kimura 2-parameter method (Kimura, 1980). Node statistics
corresponding to less than 50% bootstrap values were hidden to minimize clutter. Reference sequences are in italics
40
4.2.1.2 Phylogenetic Analysis of the Pol RT sequences
From the phylogenetic approach (Figure 4.2) most (30/41; 73.2%) of the sequences
clustered with subtype A1 reference strains. One isolate, MLD245, was orthologous to
A2D reference sequence from Kisii and was a likely recombinant of subtypes A and D
viruses. One isolate clustered with A2 references sequences. Overall, 32/41 (78%) of all
the isolates clustered with subtype A reference strains and were assigned as A viruses on
the pol-RT gene by phylogeny. Another isolate, MLD548 did not cluster with any
specific reference sequence and was unassigned by this method. KAH004 clustered
together with subtype H reference sequence, which was unusual, and could be a
recombinant strain. Subtype C accounted for 7.3% of the sequences (3/41) while
Subtype D accounted for 9.8% of the sequences (4/41).

41
Figure 4.2: Phylogenetic Relationships of HIV-1 envelope pol RT gene isolates
The evolutionary history was inferred using the Neighbor-Joining method. The evolutionary distances were computed
using the Kimura 2-parameter method (Kimura, 1980). Branches corresponding to partitions reproduced in less than
50% bootstrap replicates were collapsed and all positions 42with less than 50% site coverage were eliminated. Node
statistics corresponding to less than 50% bootstrap values were hidden to minimize clutter. Reference sequences are in
bold italics and filled circle nodes.
42
4.2.2 Comparative sensitivities of common genotyping tools used for HIV-1
subtyping
Multiple HIV genotyping tools are available for HIV subtype determination. The
comparative sensitivities of multiple platforms were assessed, so as to correctly assign
specific subtype to any HIV isolate. Four platforms were used for envelope gene and
seven platforms for pol-RT gene for subtype comparisons. For empirical purposes,
sensitivity was measured against phylogenetic method as a reference (and currently the
most robust) genotyping method. Of the 59 env C2V3 sequences, one sequence was not
long enough to be determined by all the methods and was excluded from the analysis.

For this purpose, 58 C2V3 and 41 Pol RT sequences were analysed using the respective
platforms and the results compared for concordance with subtype assignment based on
phylogenetic method. Concordance was described respectively as being either complete,
partial or none concordant if the result obtained using the automated tool was similar,
partly similar or completely different from that obtained by phylogeny respectively
(Figure 4.2). Of the 58 C2V3 sequences, NCBI reported the proportions of subtypes A1,
A2, C, D and recombinants as 23.7%, 0%, 5.1%, 0% and 71.2% respectively compared
against 73.2%, 4.9%, 9.1%, 9.1% and 0% shown by phylogeny (Table 4.3). In terms of
concordance, these translated into 29.3% (17/58), 65.5% (38/58) and 5.2% (3/58)
complete, partial and none concordance respectively. Using the COMET platform,
complete, partial and none concordance was found to be 79.3%, 13.8%, and 6.9%
respectively. REGA on the other hand reported complete concordance for 41/58 of the
samples (70.7%) and 17/58 (29.3%) as none concordant; with HIV1-A, C, D and
unassigned subtypes being 57.6%, 6.8%, 8.5%, 27.1% respectively (Table 4.3). For
JPHMM subtyping tool, complete and partial concordance was 84.5% (49/58) and
15.5% respectively; with HIV1-A1, A2, C, D and recombinants being 61.0%, 6.8%,
8.5%, 8.5% and 15.2% respectively. Through this comparative subtyping analysis
approach using different tools, JPHMM was found to be most in agreement with the
standard phylogenetic method in assigning HIV subtypes.

43
Table 4.3: Comparative concordance of various subtyping tools for assignment of
HIV-1 env C2V3 subtypes
%, (N)
NCBI COMET REGA JPHMM

Y- 29.3, (17) Y- 79.3, (46) Y- 70.7, (41) Y- 84.5, (49)


P- 65.5, (38) P – 13.8, (8) P – 0 (0) P- 15.5, (9)
N- 5.2, (3) N - 6.9, (4) N- 29.3, (17)
Key: Y= Complete concordance; P= Partial Concordance; N= Not concordant. Concordance is based on
phylogenetic method of subtyping as reference or standard method

Similar analytical approach was adopted for the pol-RT gene. For the 41 gene-specific
sequences, complete, partial or none concordance was respectively, 17.1%, 75.6% and
7.3% for the NCBI tool (Table 4.4); 46.3%, 36.6%, and 17.1% for Stanford; 75.6%,
2.4%, 22% for REGA;65.9%, 19.5% and 14.6% for JPHMM; 82.9%, 9.8% and 7.3% for
Geno2Pheno;75.6%, 19.5% and 4.9% for COMET and 56.1%, 29.3% and 14.6% for
SQUEAL (Table 4.4). NCBI reported more CRFs than all other subtyping tools while
REGA failed to identify recombinants (Table 4.4), with most unidentified sequences
reported as complex (NA). Stanford reported higher percentages of CRF_01AE,
majority of which were subtype A by other methods, and also assigned a pure subtype B
that was an A2B recombinant by JPHMM (Appendix 4). In order to have a standard
platform for the assigning of HIV-1 subtypes, JPHMM was used for subsequent analysis
of both the pol RT and env gene.

44
Table 4.4: Comparison of concordance for various subtyping tools for the pol RT
gene
NCBI STANFORD REGA JPHMM G2P COMET SQUEAL
N, (%) N, (%) N, (%) N, (%) N, (%) N, (%) N, (%)

Y-7, (17.1) Y-19, (46.3) Y-31, (75.6) Y-27, (65.9) Y-34, Y-31, Y-23,
(82.9) (75.6) (56.1)

P-31, P-15, (36.6) P-1, (2.4) P-8, (19.5) P-4, P-8, P-12, (29.3)
(75.6) (9.8) (19.5)
N-3, (7.3) N-7, (17.1%) N-9, (22) N-6, (14.6) N-3, N-2, (4.9) N-6, (14.6)
(7.3)
Key: Y= Complete concordance; P= Partial Concordance; N= None concordance

4.2.3 Genetic Heterogeneity of HIV-1 Pol RT gene from different blood


compartments
In total, 19 sequences were generated from cellular DNA samples that had undetectable
viral load and up to a maximum of 1000 HIV-1 RNA copies/ml blood. Subtyping was
done using JPHMM. Out of these, 73.7% (14/19) were subtype A1, 10.5% (2/19) were
subtype D, 5.3% (1/19) was subtype C and 10.5% (2/19) were recombinants (Table 4.5).
A total of 22 patient RNA samples were sequenced from plasma samples with viral load
above 1000 RNA copies/ml blood. Ten out of 22 (45.5%) were found to be subtype A1,
18.2% (4/22) subtype D, 4.5% (1/22) subtype C and 31.8% (7/22) were recombinants
(Table 4.5).
The recombinants obtained from cell-free plasma compartment were A1H (n=1), A1D
(n=1), A1C (n=2,), A2B (n=1), A1K (n=1) and DKD (n=1) and those obtained from
cellular blood compartment were A1A2 (n=1), A1C (n=1). HIV pol RT gene was more
diverse in the cell-free compartment than in the cellular compartment. Overall, there
were more recombinants in the cell free compartment (31.8%) compared to 10.5% in the
cellular compartment (Table 4.5).

45
The trend in distribution showed fewer subtype A strains (45.5%) in cell-free
compartment compared to cellular compartment which had 73.7% of subtype A isolates
(Table 4.5). There was a larger percentage of Subtype D in the cell-free compartment
(18.2%) compared to the cellular compartment (10.2%) (Table 4.5).

Table 4.5: Distribution and compartmentalization of HIV-1 Pol-RT subtypes


Subtype Frequency in cellular Frequency cell-free blood
blood DNA, N (%) RNA, N (%)
A (A1) 14 (73.7) 10 (45.5)
D 2 (10.5) 4 (18.2)
C 1 (5.3) 1 (4.5)
All recombinants *2 (10.5) **7 (31.8)
Total 19 (100) 22 (100)
Key:*A1C, n=1; A1A2, n=1. **A1C, n=2; A1H, n=1; A2B, n=1; A1K, n=1; DKD, n=1; A1D, n=1.

4.2.4 Genetic heterogeneity of HIV-1 C2V3 envelope gene from different blood
compartments
Similar analyses were conducted for the 59 envelope sequence isolates to assess
compartmentalization of viral genetic variants (Table 4.6). Out of the 42 isolates from
the cellular compartment, 57.1% (24/42) were subtype A1, 4.8% (2/42) were subtype
A2, 11.9% (5/42) were subtype D and 11.9% (5/42) were subtype C (Table 4.6).
Recombinant viruses accounted for 14.3% of the envelope DNA-derived virus strains
with 7.1% (3/42) being A1A2, 4.8% or 2/42 being A1H, and 2.4% (1/42) being A1D.
From cell-free plasma compartment, 70.6% (12/17) were pure envelope subtype A1 and
11.8% subtype A2 (2/17) (Table 4.6). The recombinants obtained from cell free
compartment accounted for 17.6% (3/17) and included A1D (2/17) and A1A2D (1/17))
(Table 4.6. No pure subtypes D or C were identified based on envelope C2V3 gene from
cell free RNA.

46
Overall, there was a higher percentage of subtype A in the cell free compartment
(82.4%) compared to the cellular compartment (61.9%) (Table 4.6). There was a small
difference in the percentage of recombinants in the cellular compartment (14.3%)
compared to the cell-free compartment (17.6%) (Table 4.6).

Table 4.6: Distribution and compartmentalization of envelope C2V3 subtypes


Subtype Frequency in cellular blood Frequency cell-free blood
DNA, N (%) RNA, N (%)
A (A1,A2) 26 (61.9) 14 (82.4%)
D 5 (11.9)
C 5 (11.9)
All recombinants *6 (14.3) **3 (17.6)
Total 42 (100) 17 (100)
Key: *A1A2, n=3; A1H, n= 2; A1D, n= 1. **A1D, n= 2; A1A2D, n= 1

4.2.5 Comparative distribution of HIV genetic variants between blood


compartments.
From the analyses reported in sections 4.2.3 and 4.2.4, the cellular compartment had
more of the pol-RT subtype A (73.7%) than envelope subtype A (61.9%) - Table 4.7.
The pattern was reversed for the plasma compartment which had more of the envelope
C2V3 (82.4%) than Pol-RT (45.5%) subtype A viruses (Table 4.7). Of the 61 sequences
from the cellular compartment 40 (65.6%) were subtype A (A1, 62.3%; A2, 3.3%), 9.8%
subtype C, 11.5% subtype D and 13.1% recombinants as compared to 61.5% subtype A
(A1, 56.4%; A2, 5.1%), 2.6% of C, 10.3% of D and 25.6% of recombinants in the cell-
free compartment (Table 4.7). No envelope-based subtypes C and D were identified
from plasma compartment. The plasma compartment carried more recombinant pol-RT
(31.8%) and envelope C2V3 (17.6%) viruses than the proportion of recombinants found
in the cellular compartment of similar viral gene segments (Table 4.7). Majority of the

47
subtype C isolates were dominantly identified in envelope sequences and from the
cellular compartment (Table 4.7).

Table 4.7 Comparative frequency of subtypes by genetic region and compartment

Cellular DNA, N (%) Cell-free blood RNA, N (%)


Subtype Pol-RT Env C2V3 Total N Pol-RT Env Total
(%) C2V3 N(%)
A 14 (73.7) 26 (61.9) 40 (65.6) 10 (45.5) 14 24 (61.5)
(82.4)
D 2 (10.5) 5 (11.9) 7 (11.5) 4 (18.2) 4 (10.3)
C 1 (5.3) 5 (11.9) 6 (9.8) 1 (4.5) 1 (2.6)
All 2 (10.5) 6 (14.3) 8 (13.1) 7 (31.8) 3 (17.6) 10 (25.6)
recombinants
Total 19 (100) 42 (100) 61(100) 22 (100) 17 (100) 39 (100)

4.2.6 Subtype Distribution by County


HIV has been shown to be distributed variably across different geographical settings
(see Table 2.1). This section describes the distribution of the 81 unique virus isolates
across the six Counties of Kenya, which data is part of two publications (Kitawi et al.,
2015; Nzomo et al., 2015). Of all samples qualifying for this analysis, 32% were from
Nyanza (Kisumu and Homa Bay Counties), 23.7% from Kajiado County, (Kitengela),
6.8% from Kiambu, 11.9% from Malindi and 25.4% from Nakuru County (Naivasha).
Because of differences in sample size between counties, subtype variability is assessed
within each county and not between county to avoid bias. Table 4.8 summarizes the
distribution of HIV C2V3 genotypes within each of the 6 Counties. When the JPHMM
subtyping tool was used, 67.8% of all subtypes were HIV-1A, 8.5% were subtype C,
8.5% were subtype D and 15.2% were recombinants (Table 4.8).

48
In Nyanza, of all the circulating genotypes, 68.4% were HIV-1A, 10.5% HIV-1C, 15.8%
HIV-1D while recombinants were 5.3%. From Kitengela in Kajiado county, 57.1%
were subtype A, 14.3%subtype C, 28.6% were recombinants while there was no subtype
D. Kiambu had too few samples (n=4) to be representative (Table 4.8). However, 75%
of these were subtype A and 25% were recombinants. Malindi in Kilifi County also had
very few samples with 71.4% being subtype A and 28.6% being recombinants. A larger
sample size from these counties would probably have changed the pattern seen.
Naivasha in Nakuru County had most of the samples being subtype A (73.3%), 6.7%
subtype C, 6.7% recombinants and 13.3% subtype D (Table 4.8).
Although sample distribution was not even across the sites, the data suggests that
Kitengela in Kajiado County and Malindi in Kilifi County may carry the highest
prevalence of recombinant envelope viruses, while Kiambu and Nakuru suggest a
similar trend that may need corroborating with larger sample size.

Table 4.8: Variability of C2V3 HIV genotypes within a given geographic locations
across Kenya
Number (%) in the region
A (A1&A2) C D Recombinants Total
Nyanza* 13 (68.4) 2 (10.5) 3 (15.8) 1 (5.3) 19 (100)
Kitengela 8 (57.1) 2 (14.3) 4 (28.6) 14 (100)
Kiambu 3 (75) 1 (25) 4 (100)
Malindi 5 (71.4) 2 (28.6) 7 (100)
Naivasha 11 (73.3) 1 (6.7) 2 (13.3) 1 (6.7) 15 (100)
Total 40 (67.8) 5 (8.5) 5 (8.5) 9 (15.2) 59 (100)
Key: * Ahero and Ndhiwa

The distribution of pol-RT subtypes was similarly assessed within Counties. Of all
qualifying samples for the pol RT region, 9.7% of the samples were from Nyanza, 22%
from Kitengela in Kajiado county, 12.2% were from Kiambu, 29.3% from Malindi in
Kilifi county and 26.8% from Nakuru Naivasha in Nakuru county. Table 4.9 shows the

49
variability of subtypes within the counties. The four isolates from Nyanza, were evenly
distributed by genotype at 50% subtype A and 50% recombinant strains. Kitengela in
Kajiado county had 77.8% subtype A, 11.1% subtype C and 11.1% subtype D
respectively (Table 4.9). No reverse transcriptase (RT) recombinants were found among
the 9 isolates from Kajiado. Malindi in Kilifi county had 25% subtype A1 and D, 8.3%
subtype C and 41.7% RT recombinants. Naivasha in Nakuru county had majority of the
samples (81.8%) being subtype A1, 9.1% being subtype D and 9.1% being recombinants
(Table 4.9). The overall pattern was that there were more recombinant isolates for the
pol RT than envelope genes.

Table 4.9: Variability of Pol RT HIV genotypes within a given geographic locations
across Kenya
Number (%) in the region
A1 C D Recombinants Total
Nyanza* 2 (50) 2 (50) 4 (100)
Kitengela 7 (77.8) 1 (11.1) 1(11.1) 9 (100)
Kiambu 3 (60) 1 (20) 1 (20) 5 (100)
Malindi 3 (25) 1 (8.3) 3 (25) 5 (41.7) 12 (100)
Naivasha 9 (81.8) 1 (9.1) 1 (9.1) 11(100)
Total 24 (58.5) 2 (4.9) 6 (14.6) 9 (22) 41(100)
*Sequences from Ahero and Ndhiwa

4.3 Co-receptor Usage


Two of the 59 sequences did not span the V3 region and were thus deemed too short for
coreceptor analysis. Four of the 59 (including 2 with short V3 region), had C2V3 region
too short for phylogenetic subtyping, but were still able to be subtyped based on
JPHMM and other online bioinformatics platforms. Of the 57 sequences with complete
V3 region, 34 were subtype A1, 9 were recombinants of A1, 4 were A2, 5 were subtype
C and 5 were subtype D.

50
Three different FPR cut-offs (5%, 10% and 20%) were applied as described under
methods. Forty three of the 57 sequences (75%) were CCR5 (R5) tropic and 14 (25%)
were CXCR4 (X4) tropic viruses at FPR10. No dual tropic (R5X4) virus isolates were
found using the Geno2Pheno phenotyping algorithms. For all three FPR cut-off
algorithms, there was a significant relationship between viral subtype and viral tropism
(χ2 p=0.037; p=0.016 and p=0.005 for FPR5, FPR10 and FPR20 respectively).
Clustered by viral subtype, approximately 80% of subtype A1 sequences were R5 tropic,
against approximately 20% X4 viruses. Similarly, a larger proportion (approximately
75%) of A1 recombinant viruses were R5 tropic. Relative to subtype A1 viruses, there
were however, comparably fewer subtype A2 viruses (n=4), 50% of them being either of
X4 or R5 phenotype. None of the subtype C viruses were of X4 phenotype using all
three FPR algorithms, compared to nearly all (80% at FPR10 and 100% at FPR20) of
subtype D viruses being found to be X4-tropic. These analyses suggested a trend
whereby more of the pure subtype A1 were likely to be assigned as R5 tropic at lower
FPR than were assigned as X4 viruses, while more of the pure subtype D isolates tended
to be assigned as X4 tropic at higher FPR. Tropism of pure subtype C and A2 viruses
appeared unaffected across FPR algorithms, while that of recombinant strains either
varied minimally or not at all. Table 4.10 shows the distribution of viral tropism across
HIV subtype variants at the 3 FPR cut-offs.

51
Table 4.10: The distribution by number and (proportions), of viral tropism across
HIV subtype variants.
Viral Tropism at FPR cut-off (N, %)
FPR, 5% FPR (10%) FPR (20%)
HIV-1 CCR5 CXCR4 CCR5 CXCR4 CCR5 CXCR4 TOTAL
Subtype
A1 30 (88.2%) 4 (11.8%) 28 (82.4) 6 (17.6) 26 (76.5) 8 (23.5) 34 (100)
*A1r. 7 (77.8) 2 (22.2) 7 (77.8) 2 (22.2) 6 (66.7) 3 (33.3) 9 (100)
A2 2 (50) 2 (50) 2 (50) 2 (50) 2 (50) 2 (50) 4 (100)
C 5 (100) 0 (0) 5 (100) 0 (0) 5 (100) 0 (0) 5 (100)
D 2 (40) 3 (60) 1 (20) 4 (80) 0 (0) 5 (100) 5 (100)

Total 46(81) 11 (19) 43 (75) 14 (25) 39 (68) 18 (32) 57 (100)

4.4 Patterns of Potential N-Linked Glycosylation Sites (PNGs)


The subtype of 55 out of 59 sequences could be defined phylogenetically (Kitawi et al.,
2015). These were the sequences that were examined for the number and pattern of N-
linked glycosylation sites (PNGs) as described under methods. In terms of specific
amino acids (A.A), the largest number of virus isolates were glycosylated at asparagine
(N) position 277 (N277, 73%) and at N302 (71%) (Table 4.11). The proportion of
sequences with PNGs at specific amino acid positions was 80% (44/55) for N262 and
98.2% (54/55) for N277. The rest were: N296 (38/55, 69.1%), N302 (54/55, 98.2%),
N337 33/55 (60%), N345 (40/55, 72.7%), N366 (41/55, 74.5%), N399 (43/55, 78.2%)
and N408 (40/55, 72.7%). These patterns remained largely true when data was further
disaggregated by nucleic acid source as either being derived from DNA or RNA material
and are presented in Table 4.11.

Nucleic Acid (NA) source material was used as a proxy for viral compartmentalization
in blood, with plasma-derived RNA isolates being representative of extracellular blood

52
compartment and PBMC-derived DNA isolates representing cellular compartment. For
the 55 sequences, the total number of PNGs considering all possible patterns was 615
(mean, 11.18, standard error –SE -, 0.274). Of these, 403 were of NXT type (mean, 7.33,
SE 0.239) while 205 were of NXS type (mean, 3.73, SE 0.167) with only 7 being of the
contiguous NNXS(T) pattern (mean, 0.13, SE 0.052). Table 4.12 summarizes the mean
PNGs of isolates clustered according to subtype, NA source and viral tropism.

53
Table 4.11: The distribution and clustering of specific amino acid PNG sites
according to viral tropism and NA source material
Number of isolates; % possessing amino acid PNG at specified site
Tropism N262 N27 N296 N30 N337 N34 N366 N399 N40 Total
7 2 5 8
DNA source

CCR5 22; 29; 20; 28; 18; 22; 26; 24; 24; 29;
66.7 72.5 71.4 71.8 72 73.3 81.3 68.6 72.7 72.5
CXCR4 11; 11; 8; 11; 7; 28 8; 6; 11; 9; 11;
33.3 27.5 28.6 28.2 26.7 28.7 31.4 27.3 27.5

All DNA 33; 40; 28; 39; 25; 30; 32; 35; 33; 40;
100 100 100 100 100 100 100 100 100 72.7

RNA source
CCR5 9; 81.8 12; 9; 90 13; 7; 8; 80 7; 7; 5; 13;
85.7 86.7 87.5 77.8 87.5 71.4 86.7

CXCR4 2; 18.2 2; 1; 10 2; 1; 2; 20 2; 1; 2; 2; 23.3


14.3 13.3 12,5 22.2 12.5 28.6

All RNA 11; 14; 10; 15; 8; 10; 9; 8; 7; 15;


100 100 100 100 100 100 100 100 100 27.3

Total
CCR5 31; 41; 29; 41; 25; 30; 33; 31; 29; 42;
70.5 75.9 76.3 75.9 75.8 75 80.5 72.1 72.5 76.4
CXCR4 13; 13; 9; 13; 8; 10; 8; 12; 11; 13;
29.5 24.1 23.7 24.1 24.2 25 19.5 27.9 27.5 23.6
All 44; 80 54; 38; 54; 33; 40; 41; 43; 40; 55;
98.2 69.1 98.2 60 72.7 74.5 78.2 72.7 100%
Key: NA – Nucleic Acid. R5 or X4 tropism on this analysis is based on false positive rate of 10%

54
Table 4.12: Mean PNGs for 55 isolates by subtype, nucleic acid source and tropism
Mean PNGs (Standard Error)
NXS NNXS(T)
All Patterns NXT Pattern Pattern Pattern

Subtype A1 11.18 (0.386) 7.24 (0.346) 3.79 (0.214) 0.15 (0.075)

A1 R 10.57 (0.685) 6.71 (0.36) 3.71 (0.474) 0.14 (0.147)

A2 10.75 (0.629) 7.75 (0.854) 3.00 (0.913)

C 11.2 (0.663) 7.4 (0.678) 3.80 (0.583)


D 12.4 (0.748) 8.4 (0.40) 3.80 (0.374) 0.2 (0.2)

Nucleic DNA 11.6 (0.288) 7.68 (0.252) 3.85 (0.198) 0.08 (0.042)
acid
source RNA 10.07 (0.565) 6.40 (0.505) 3.40 (0.306) 0.27 (0.153)

Tropism CCR5 11.02 (0.331) 7.21 (0.282) 3.64 (0.192) 0.17 (0.067)
CXCR4 11.69 (0.429) 7.69 (0.444) 4.00 (0.340)

Total 11.18 (0.274) 7.33 (0.239) 3.73 (0.167) 0.13 (0.052)

Clustered by blood compartment, there were 12 PNGs for each viral isolate derived from
DNA (cellular) compartment compared to 10 PNGs from each isolate of RNA
(extracellular) material. DNA derived isolates were still the most glycosylated at NXT
sites. Analysis of variance (ANOVA) was next conducted to compare mean values of
the specific PNG patterns across NA source. This analysis revealed a significantly
different pattern and density of PNG between cellular and extracellular viral isolates for
the NXT glycosylation pattern (p=0.016), and for all the patterns combined (p=0.011) -
see Figure 4.3 below. Moreover considering specific amino acid PNG sites, there was

55
still a significant difference in glycosylation between cellular and extracellular isolates at
A.A positions N399 (p=0.006) and N408 (p=0.007).

Figure 4.3: Proportion of sequences containing PNGs at specific amino acid


positions. Figure 1A: all isolates; Figure 1B: isolates derived from DNA; Figure 1C:
isolates derived from RNA.

When all the possible PNG patterns were considered, the mean PNGs for A1 was 11.18
(SE 0.386), A1 recombinants was 10.57 (SE 0.685), A2 was 10.75 (SE 0.629), C was
11.20 (SE 0.663) and D was 12.40 (SE 0.748). Thus subtype D isolates had the largest
number of PNGs per isolate while recombinant strains had the least. Similarly, subtype
D was the most abundantly glycosylated at NXT sites (mean of 7.33, SE, 0.239), with
A1 strains being least glycosylated at these sites. Glycosylation density was comparable
at NXS sites for subtypes A1 (mean 3.79; SE 0.214), subtype C (mean, 3.80; SE 0.583)
and subtype D (mean, 3.80; SE 0.374) but much less for A1 recombinants (mean

56
3.00,SE 0.601) and subtype A2 (mean, 3.00; SE 0.913). Glycosylation at the contiguous
NNXS(T) sites was the least common and was observed at a mean occurance of 0.15 per
isolate for A1 viruses, 0.14 (SE 0.14) for A1 recombinants, and 0.2 (SE 0.2) for D
viruses. None of the subtype C or A2 isolates were glycosylated at the NNXS(T) site.
Table 4.12 shows the details for the average number of pattern-specific PNGs
disaggregated by viral subtype, tropism and source material. An ANOVA test to
compare mean PNGs for the different subtypes at specific amino acid position yielded
significant results for position N296 (p=0.021), N302 (p=0.034) and N366 (p=0.016).

57
CHAPTER 5

DISCUSSION

5.1 Patient Characteristics


This study focused on investigating distribution and genetic diversity of HIV-1 in
Homa-Bay, Kisumu, Kiambu, Nakuru, Kajiado and Kilifi counties of Kenya. Patients
were recruited from six facilities located in these counties and sequence information was
obtained from 81 patients. By gender, 32.1% (26/81) of the patients were males, 64.2%
(52/81) were females and 3.7% (3/81) were not recorded. The Ministry of Health Kenya
County HIV profiles showed a higher prevalence of HIV in Kenya among women
(7.6%) than men (5.6%) (NACC., 2014). The distribution of the patients by age was
7.4% (6/81) below 25 years old, 33.3% (27/81) between 25 and 35 years, 44.5% (36/81)
between 36 and 50 years, 11.1% (9/81) above 50 years and 3.7% (3/81) were not
recorded.
The patients had been on treatment for a median length of 40 months. CD4 T-cell count
was less than 350 cells/mm3 for approximately 50% of the patients, with 27% having a
CD4 count greater than 500. About 60% of the patients had their viral load (VL) below
detectable levels (39 HIV-1 RNA copies/ml blood). The Guidelines for antiretroviral
therapy in Kenya indicate that the viral load should be below detectable levels within six
months of ART (NASCOP, 2011). Although there is a sharp decline in viral load within
6 months of treatment, the CD4 count increases slowly over time (NASCOP, 2011) and
this would explain why 50% of the patients had their CD4 count below 350 despite a
higher percentage (60%) having their viral load below detectable levels. HIV infection
affects more women (59.1%) than men (40.9%) in Kenya (NACC & NASCOP, 2012),
and this could be one reason why more women were enrolled in the study than men.
About 9.9% of the patients were not on ART. Those on ART, were receiving the
following treatment regimens (percentages given based on total number of patients):
1.2% on ABC+3TC+NVP, 33.3% on AZT+3TC+NVP/EFV, 19.8% on

58
D4T+3TC+NVP/EFV and 24.7% on TDF+3TC+NVP/EFV, with NVP and EFV being
used as alternate non-nucleoside reverse transcriptase inhibitors (NNRTI). ART regimen
for 11.1% of the patients was not recorded (NR). The recommended first line ART in
Kenya is AZT+3TC+NVP/EFV or TDF+3TC+NVP/EFV(NASCOP, 2011). Although
the recommended treatment was the most common among patients receiving ART, the
continued use of D4T in ART was contrary to existing WHO recommendation for D4T
phase-out due to its association with toxicity and intolerance (WHO, 2009.).

5.2 HIV-1 subtype diversity


Subtype diversity is influenced by multiple factors, including cross-border demographic
patterns that affect transmission and gene flow (Hemelaar et al., 2011). A number of
studies have reported subtype A as the predominant subtype in Kenya (Table 2.1). In
this study, subtype A was still the predominant subtype, majority of these being sub-
subtype A1.
A number of subtype A1 sequences clustered with reference sequence from Uganda,
which shares the border with Kenya to the west (for C2V3 region, KAH008, MLD258,
KHC059, KHC069, KMB154, KAH203; and MLD198, KMB160 for Pol RT region).
The clustering observed for sequences from Kisumu (prefix -KAH) with reference
sequences from Uganda is probably due to the fact that Kisumu is located almost at the
border with Uganda. A pol RT sequence from Malindi, MLD040, clustered with an A1
sequence from Senegal. Malindi being a tourist town, this could have been due to
migration. The subtype C sequences clustered with reference sequences from South
Africa (KHC052 and NVS148 for C2V3) and Ethiopia (KHC040 for both C2V3 and Pol
RT). South Africa and Ethiopia have predominantly subtype C (R. Lihana et al., 2012;
Tully & Wood, 2010). MLD021 for Pol RT and NVS152 for C2V3 clustered with
subtype D reference sequences from Uganda and HND164 with a reference sequence
from the Democratic Republic of Congo. For the Pol RT region, one of the sequences,
KAH004 clustered with a subtype H from Cameroon which is an unusual subtype for
Kenya. However, this sequence was comparatively short, which may reflect a
compromised sequence quality and integrity and consequently, a biased subtype

59
assignment on the phylogenetic tree. A number of sequences were orthologous and in
some cases had similar branch lengths showing that these sequences were
phylogenetically closely related despite coming from different counties. The orthologous
sequences for the C2V3 region included KAH009 and NVS095; KAH001 and NVS121;
HND046 and KHC013; KAH157 and KHC050; KHC052 and NVS148; KAH010 and
KHC064, while those for the Pol RT region included KHC163 and NVS069; KMB145
and MLD545; KHC045 and KMB067; KMB077 and NVS058. These similarities
suggest restricted evolutionary trend and virus transmission within the population across
the country. A study with a larger sample size will probably bring out these dynamics to
a greater extent.

It was hypothesized that the diversity of HIV in Kenya could be on the rise due to
increasing demographic heterogeneity, human migration and ease of travel within
Kenya; increasing influence of antiretroviral pressure in addition to generalized viral
evolution. To capture this diversity, the plasma and cellular viruses were both assessed.
It has been shown that HIV subtypes vary between various compartments, with
suggestion that inter-compartment recombination may anchor evolutionary mechanism
that helps shape intra-host diversity of HIV-1 env gene in natural infection (Brown et
al., 2011). Compartmental differences in genetic diversity were observed in this study as
well, particularly with the pol RT sequences. The cellular compartment had 10.5% of the
sequences being recombinants while the cell-free compartment had 31.8% of the
sequences being recombinants. This difference was less remarkable for the C2V3
sequences, where recombinant viruses were 14.3% in the cellular compartment against
17.6% in the cell-free compartment. The pol RT region of HIV-1 is the target for current
antiretroviral drugs (NASCOP, 2011). Constant exposure to these drugs and lack of
adherence and compliance is associated with drug resistance mutations, which can
further enhance variability (Ochieng et al., 2015). Zidovudine, a Nucleoside Reverse
Transcriptase Inhibitor (NRTI) for example, can enhance the mutation rate of HIV-1 by
a factor of 7 for each replication cycle, and HIV-1 variants harbouring Zidovudine

60
resistant reverse transcriptase can have a mutation rate that is 3 fold that of the wild
virus type (Martinez-Picado & Martinez, 2008).

Production of variants that are endowed with superior fitness that arises due to
recombination has been observed in cases of dual or triple infection of a patient
(Templeton et al., 2009; Van der Kuyl & Cornelissen, 2007). In this study, 19 isolates
were sequenced for both the pol RT region and the C2V3 region. Using JPHMM, it was
found that the subtype was discordant for the pol RT and C2V3 region (polRT:C2V3), for
7 isolates KAH004 (A1D:A1), KAH010 (A1:A1D), KHC045 (A1:A1H), KHC048
(A1:A1A2), MLD011 (D:A1D), MLD245 (A2B:A2), NVS032 (A1:A1H). Using
phylogeny, subtype was discordant for 3 isolates KAH004 (H:A1), MLD011 (D:A1) and
MLD 245 (A2D:A2). Phylogenetic method is not powered to detect recombinant strains,
and there is a possibility that the unassigned isolates or strains in unusual clusters are
recombinant viruses. Recombination observed in these instances, especially using
phylogeny, is highly indicative of dual or triple infection in these patients.
Recombination between variants that are highly similar occurs more frequently than
with variants that are distant (Motomura, J. Chen, & Hu, 2008). Malindi, a town at the
Coast of Kenya whose main economic activity is Tourism, was found to have many of
these recombinants (prefix of sequence ID - MLD). Hue reported a similar pattern of
recombinants at Kilifi, a town also located at the Coast of Kenya and about 100km from
Malindi (Hué et al., 2012).The recombinants they found in Kilifi using various methods
include A1-AE, A1-A2, A1-A2-B, A1-A2-D, A1-C, A1-D, A2-B, A2-D, AE-C, B-C, C-
D. It is highly probable too, that such variants are spread through intravenous drug use
(IDU). The proportion of HIV infections linked to IDU at the Coast of Kenya is 17%
compared to 4-5% in the whole country (Nieburg & Lisa, 2011). The high proportion of
recombinants (in the pol RT region) in Malindi as compared to the other counties
correlates well with what Hue found in Kilifi (Hué et al., 2012).

61
The cellular compartment had a greater proportion and variety of pure subtypes than the
acellular compartment, this observation being more remarkable with the env region than
the pol RT region.

5.3 Co-receptor Usage


The analysis of C2V3 sequences generated from this study using the Geno2pheno
platform revealed that 75% of the viruses used CCR5 as their co-receptor of choice (R5-
tropic) while 25% used CXCR4 as co-receptor (X4-tropic) for viral entry to CD4 T-cells
at FPR10. No dual tropic sequences were found by the method used, which may be due
to lack of sensitivity of the method to pick dual tropism. Site differences in tropism was
not analysed due to differences in sample size that could bias the result. Subtype
differences in co-receptor usage have been reported in different studies though few
coreceptor studies have been done in Kenya. A few of the local studies with varied
numbers of subjects have found majority (73%-84%) of HIV-isolates to be CCR5 tropic,
although comparatively fewer samples were assessed (R.W. Lihana et al., 2009b;
Nyamache, Muigai, Ng'ang'a, & Khamadi, 2013; Wambui et al., 2012) . In the current
study, using a FPR of 10%, 81.8% of the A1 sequences were R5-tropic and 19.2% were
X4-tropic sequences. These findings are in accord with the general trajectory of HIV-1
subtype A co-receptor tropism from the local population.

Subtype C has been shown to preferentially use CCR5 and to rarely induce syncytia
(Abraha et al., 2009; Bessong et al., 2005). The current study found that all subtype C
isolates were R5-tropic. CXCR4 use is generally associated with a more rapid decrease
of CD4 counts and therefore faster disease progression, with subtype D viruses
preferentially being X4-tropic (Noah Kiwanuka et al., 2008; Wambui et al., 2012). The
study by Kiwanuka and colleagues attributed faster rate of disease progression in
subtype D to a higher frequency of syncytium formation and tropism for CXCR4 than
other subtypes. In the present study, a greater proportion i.e. 80%, of subtype D
sequences were X4-tropic at FPR10 while at FPR20, all isolates were X4-tropic.

62
5.4 Number of Potential N-Linked Glycosylation (PNG) Sites
Glycans play various roles in the infective cycle of the virus. Any change in the
glycosylation sites can affect protein folding and conformation. The envelope of HIV-1
becomes coated with so many glycans that the virus can become „invisible‟ to (protected
from) the immune response of the host (Poon et al., 2007). In order to assess the
relationships among PNG, HIV subtype and viral tropism, the number and proportion of
PNGs were compared between coreceptor tropism (CCR5 versus CXCR4 tropic) and
between the different genetic source materials (DNA versus RNA).

Compared by different blood compartment and genetic material, this study found a
higher and significantly different frequency of PNGs in DNA (cellular blood) than RNA
(cell-free blood). The difference was more exemplified by the NXT pattern of PNGs
(p=0.016). It would be expected that RNA isolates would be more glycosylated as a way
of counteracting the more intense selection pressure seen in this compartment due to
host defense mechanisms or pressure from anti-retroviral therapy (Ho et al., 2008). A
possible explanation for the deviation from this expectation could be subtype
differences. The study by Ho and colleagues comprised mainly subtype B viruses while
majority of isolates in this study were of subtype A.

Comparing PNGs by tropism, X4 tropic viruses were more glycosylated in this study
than the R5-tropic viruses both for the NXT or NXS pattern. Existing evidence from
mainly subtype B and C viruses suggest that R5-tropic strains tend to be more
glycosylated than their X4 counterparts (Kalinina et al., 2013; Zhang et al., 2004). The
deviation from literature can be explained by the fact that majority of HIV-1 strains in
Kenya and as shown from this study, are subtype A1. All the other subtypes in this study
had more PNGs on average for the R5-tropic virus than the X4-tropic virus.
PNGs among other genetic and structural characteristics, have been associated with
enhanced transmission virus (C.B. Wilen et al., 2011). Comparing PNGs by subtype
variations, subtype A was found in a separate study to have greater selection for viruses

63
with shorter V1-V2 loops and fewer PNGs (Chohan et al., 2005). The R5 tropic virus is
preferentially transmitted compared to the X4 tropic virus (Pollakis et al., 2001). The
ease of transmissibility could also be enhanced for subtype A viruses (Chohan et al.,
2005), by having fewer PNGs. This study found that the R5 tropic subtype A viruses
were less glycosylated than the X4 tropic virus, a characteristic that would enhance their
transmissibility. It may be that as the disease progresses (and the tropism also shifts
from R5 to X4 tropism), the virus adapts itself to fight the pressure from neutralizing
antibodies by increasing its PNGs. X4-tropic viruses have been observed during later
stages of infection and induce synctia formation that leads to faster disease progression
(Berger et al., 1998). It would be useful to do a review of the Subtype A1 viruses in
Genbank, analyzing their N-linked glycosylation patterns and their coreceptor usage and
comparing this with subtype A2, A1 recombinants and other subtypes.
The relationship between Total PNGS, NXT, NXS patterns and coreceptor usage though
different on average was found to be not significant. The same applies with the
relationship between the PNG patterns and subtype. Some glycosylation sites have been
found to have a high frequency of glycosylation. The study also found a significant
relationship, using chi square test, between subtype and glycosylation at N296
(p=0.021), N302 (p=0.034) and N366 (p=0.016). The significance of this is yet to be
determined.

5.5 Conclusion
It can be concluded from this study that:
1. The major subtype in Kenya is still A1 although one can see different patterns in
different regions and an increasing number of recombinants.
2. Comparison between the cellular and acellular compartments indicates that there is
greater recombination in the acellular compartment and this can be explained by the
fact that there is greater immune pressure within this compartment than the cellular
compartment.
3. Seventy-five percent of the sequences were predicted to use the CCR5 coreceptor at
the FPR of 10%. There were subtype differences in the use of coreceptor with

64
subtype A1 having 80% of the sequences being CCR5-tropic, subtype A2 being
50% CCR5-tropic, subtype C being 100% CCR5-tropic and subtype D being 20%
CCR5-tropic. These differences in tropism have direct implications on disease
transmission and progression.
4. N-linked glycosylation patterns showed inter-subtype variability with subtype A1
(which were the majority of the sequences) having less PNGs for the R5-tropic
virus than the X4-tropic virus. The reverse pattern was seen for the other subtypes.
This study also showed more PNGs from the isolates obtained from DNA than
RNA for the subtype A virus.

5.6 Limitations of the study


1. In carrying out PCR, negative results were obtained severally due to contamination
of the machinery and the laboratory itself. This meant that fewer samples than
expected amplified for the required gene region.
2. In assessing coreceptor usage, phenotypic studies are more rigorous than genotypic
studies. There wasn‟t sufficient capacity at the time of this study, to carry out
phenotypic analyses.

5.7 Recommendations
1. It may be helpful, if resources are available, to derive samples from several health
facilities in the same county so as to ensure that samples obtained are more
representative of the entire population of that county. This will enable a more
comprehensive analysis of HIV-1 diversity within the county.
2. Coreceptor studies that incorporate phenotypic analysis could be done so as to
provide a more in depth characterization.
3. Carrying out an N-linked glycosylation analysis of the entire C1-V5 region of env
may yield more useful information about the interaction of the different N-glycosite
positions and subtype differences in the pattern of PNGs.

65
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78
APPENDICES

Appendix 1A: Consent Information for Study Participants (English)


Characterization of HIV Type 1 Genetic and Viral Diversity in Various Counties of
Kenya
Rose Chari Kitawi; MSc Epidemiology, Jomo Kenyatta University of Agriculture
and Technology (JKUAT)
Information to study participants

HIV can get into your blood through several ways such as when someone with the virus
gives you there blood, accidents, or during or before birth when the mother has the virus.
Anybody can get the virus! Although there are medicines to treat HIV, there is no cure.
Available medicines also do not cure and sometimes they may cause other problems.

We want to know if drugs given to you are working well or they have failed to help you.
If you accept, we will take a small amount of blood, the size of 2 full table spoons so as
to look at how the drugs work and how the virus responds to those drugs. We are not
giving you a different treatment, so you will have to continue taking the current
medicine unless your doctor makes changes

Benefits of the study

If we use your blood to know why sometimes the medicines don‟t work, we can tell your
doctor to give you better medicine and we can also use that knowledge to make better
medicines which will help many people, including yourself in the future.

Confidentiality of your identity

We will not tell anybody anything about you. Nobody will know that you gave us blood.
We will not even say something to anyone about your results.

Medical risks and problems.

79
When we take blood, you may experience some pain from the injection by needles.
Sometimes the place may swell, but that will disappear shortly. We don‟t expect you to
have any other problems because of the blood we take. But doctors will be available for
you in case you feel different from before blood was taken.

Storing your blood for future studies

We will also keep your blood to look at it later.

Exportation of your samples:

Sometimes, we may send the blood out of Kenya where additional tests may be done if
we do not have the equipment and chemicals to do it locally.

Obtaining additional information

If you have any questions at any time, please ask our Doctors and nurses and they help
you. Their numbers are below, which you can also call.

Contact: Rose Kitawi 0733432175

Basis of participation

We will only take your blood if you agree. If you say no, we will not take blood. If you
refuse, no one will know you talked to us and you will still be treated at this hospital as
usual.

Tell us if you agree or if you don‟t agree to participate in this study

I agree ................... refuse ...................to participate in this study.

Also tell us if you agree that we keep your blood to do tests later:

I agree ................refuse .............

Also tell us if you agree that we can send your samples out of Kenya to be tested:

80
I agree to ..................refuse ...................

Project Personnel to fill this section before the participant:

Unique study
No.…………………Nationality/origin..………………………………………

Age……………… …………… ………… Sex ………………...


………………………..

Occupation…………………. ………………… Locality


…………………………………..

Subject Recruited Not recruited to the study (Note: refusal by child


overrides parental/guardian consent.

81
Appendix1B: Fomu ya idhini kwa wanauhusika katika utafiti huu (Kiswahili
version)
Characterization of HIV Type 1 Genetic and Viral Diversity in Various Counties of
Kenya
Rose Chari Kitawi; MSc. Epidemiology, Jomo Kenyatta University of Agriculture
and Technology (JKUAT)
Taarifa kwa washiriki

Kuna njia nyingi ambazo unaweza kuambukuzwa ukimwi. Moja yao ni wakati wa
kuzaliwa. Watoto haswa huambukizwa wakati wa kuzaliwa. Ijapokua kuna dawa za
ukimwi, bado hakuna tibabu kamili au kinga.Virusi vinavyo ambukiza ukimwi vinaitwa
HIV. Kuna aina mbalimbali ya HIV. Kuna aina zingine ambazo ni ngumu kutibu na
huleta shida nyingi. Wakati mwingine HIV ya kawaida hubadilika na huzidi kuleta
shida.

Kupitia uchunguzi huu, tunataka kuelewa zaidi jinsi HIV inavyobadilika na kuleta shida
wakati mtu anapotumia dawa.

Tutakavyofanya Uchunguzi

Tutahitaji damu kiwango kidogo cha milliliter 10. Hiyo damu tutaipeleka kwa chumba
cha utafiti ilitupime aina ya virusi vinavyopatikana kwa damu yako.

Ni faida gani utapata kutoka kwa uchunguzi huu?

Yale matokeo tutakayopata kutoka kwa upelelezi huu yatatumika kutengeneza dawa
zilizo na nguvu ya kukabiliana na ukimwi. Matokeo hayo basi yatakuwa ya manufaa
kwako na kwa wote walioambukizwa na virusi hivi.

Je, kuna wengine watakaojulishwa kuhusu ugonjwa wako au shida ulizonazo za kiafya?

La, hatutamwelezea mtu yeyote yule kuhusu ugonjwa wako au shida zozote ulizonazo za
kiafya. Hata wanaofanya uchunguzi hawatajua jina lako kwasababu hatuandiki majina.

Kuna madhara gani kuhusianana uchunguzi huu?

82
Unaweza kuhisi uchungu kidogo tunapokutoa damu lakini hiyo ni kawaida. Ukihisi
tatizo lolote unaweza kumfahamisha daktari wakati utakopotembelea kituo cha afya.

Kuna njia zingine zaidi tutatumia damu yako?

Ukikubali, tutahifadhi damu yako kwaajili ya uchunguzi wa baadaye. Pia huenda


tukasafirisha damu yako nje ya Kenya tunapohitaji uchunguzi zaidi. Una uhuru
kukubaliana na haya au unaweza kukataa

Maelezo zaidi

Utakapohitaji kuuliza maswali wakati wowote ule, wanautafiti wako tayari kukujibu. Pia
unaweza kupiga simu wakati wowote kupitia nambari ifuatayo.

Rose Kitawi: 0733432175

Makubaliano

Tafadhalionyeshakwaalamahapachinichaguolako.

Unakubalia au unakataa kuhusika na Utafiti huu?

Ninakubali ................... Ninakataa ...................

Je unakubali kuhifadhiwa kwa damu yako?

Ninakubali ................... Ninakataa ...................

Je unakubali damu yako isafirishwe ngambo?

Ninakubali ................... Ninakataa ...................

Project Personnel to fill this section before the participant:

Unique study
No.…………………Nationality/origin..………………………………………

Age……………… …………… ………… Sex ………………...


………………………..

83
Occupation…………………. ………………… Locality
…………………………………..

Subject Recruited Not recruited to the study (Note: refusal of consent by


children 13 to 17 overrides parental/guardian consent whereas refusal by guardian/parent
nullifies child‟s consent).

84
Appendix 2A: Parental/Guardian consent for children 13-17 years (English).

About the study: We are doing a study to look at why some types of HIV cause more
problems to treat than others. If we can answer that question, we can better understand
how to treat HIV.

What about your child? We have talked to your child and explained about this study.
Your child has the right to agree or refuse to participate and that choice will be
respected. As a parent, you also have a right to refuse your child‟s participation.
Although your child‟s choice will be respected, if they agree to participate but you
refuse, we will not put them in the study.

Why are we telling you this? We want to know if you agree to have your child
participate in this study or if you do not agree. Please make your choice next each
section below

Do you agree that your child take part in this study?

I agree I do not agree

Do you agree that your child’s blood may be kept for testing later?

I agree I do not agree

Do you agree that your child’s blood sent outside Kenya to be tested?

I agree I do not agree

85
Appendix 2B: Idhini ya Mzazi wa mtoto mwenye umri wa miaka 13-17 (Kiswahili
Version).

Kuhusu uchunguzi huu


Huu uchunguzi utakagua jinsi virusi vya Ukimwi vinavyo kiuka madawa yanayo tibu
ukimwi. Mtoto wako ameombwa kuhusika na utafiti huu. Kama mzazi, una haki ya
kukataa mtoto asihusike. Tunakupatia hiyo fursa utuambie kama utamkubalia mtoto
ahusike au la. Ikiwa mmoja wenu (mtoto au wewe kama mzazi) atakataa, basi mtoto
hatahusika kamwe.
Tafadhali chagua moja kati ya chaguo mawili yanayofuata
Unakubali mtoto ahusike na uchunguzi huu?

Ndiyo, nakubali La, nakataa

Unakubali damu ya mtoto ihifadhiwe kutumika baadaye inapotakikana?

Ndiyo, nakubali La, nakataa

Unakubali damu ya mtoto itumwe ngambo ikaguliwe zaidi?

Ndiyo, nakubali La, nakataa

Project Personnel to fill this section before the participant:

Unique study No.………………… Nationality/origin..……………………………

Age……………… Sex ……………….. Occupation………………….

Locality …………………………………..

Subject Recruited Not recruited to the study (Note: refusal of consent by


children 13 to 17 overrides parental/guardian consent whereas refusal by guardian/parent
nullifies child‟s consent).

86
Appendix 3A: QUESTIONNAIRE (English version)
Unique study No.…………………… Nationality/origin..………… ………………

Age……………… Sex ………………….. Occupation………………….

Residence ………………………………….

1. Do you consider yourself as a Kenyan? Yes....... No.......


2. If No, what is your country of origin.......................?
a. How long have you been in Kenya? ..................
b. When did you arrive? ..............
c. How often do you travel back and forth across the border
3. Do you travel often to countries other than Kenya? Yes....... No.......
a. If Yes, which one(s)
b. Which country did you last visit.................when...........
4. Do you travel often to regions in Kenya outside where you live now? Yes.......
No.......
a. If Yes, which one(s)
b. Which county/region did you last visit.................when...........
5. Are you married? Yes....... No....... (if No, proceed to 6)
a. If married, do you currently live together with your spouse? Yes.....
.No.....
i. Do you consider your relationship intimate? Yes.......No.......
b. If not living together, do you ever come into contact Yes.......
No.......
i. How regular is the contact:
ii. Is it an intimate contact:
6. Do you have sex partner(s) Yes..... .No.....
a. If Yes, how many?............
b. Do you ever come into sexual contact Yes..... .No.....

87
c. If Yes, how regular?...................
7. Have you taken HIV test before? Yes..... .No.....If no, are you willing to take one?
8. Do you know your HIV status? Yes..... .No..... If no, do you want to know?
9. Does your partner/spouse know your HIV status? Yes..... .No.....
a. If No, do you want them to know? Yes..... .No.....
b. If Yes, do you need help to discuss with them your status? Yes.....
.No.....
10. Has your partner/spouse taken HIV test before? Yes..... .No.....
a. If Yes, do you know their status? Yes..... .No.....
b. If No, do you continue to have sexual contact together? Yes.....
.No.....
11. How long has it been since you first knew about your HIV status?
12. Are you taking any HIV drugs? Yes......No.....
a. If Yes, how long since you started?
b. Have you had to change the drugs because of complications?
i. If Yes how many times?
c. Have you had to stop taking the drugs temporarily for personal reasons
d. Have you ever forgotten or postponed taking the drugs
i. How often?
e. Do you take them regularly now? Yes..... .No.....
13. If not taking HIV drugs, do you know you can get them free? Yes......No.....
a. Do you want to start taking HIV drugs? Yes......No.....
14. Do you agree to extended storage of your blood/derivative samples for future
analyses? Yes......No.....
15. Is there anything you want to tell me or questions you wish to ask?

88
Appendix 3B: MAHOJIANO
Nambari ya Utafiti.…………………… Utaifa/Asili..………… …………

Umri…………… Mme/Mke ………………Kazi……………………………

Makao ………………………………….

1. Je, wewe in Mkenya? Ndio....... La.......


2. Kama si Mkenya, Nchi yako ni gani.......................?
a. Ume umeishi humu Kenya kwa mda gani? ..................
b. Tangu lini umeishi humu nchini? ..............
c. How often do you travel back and forth across the border
3. Je, wewe husafiri mara nyingi kwa nchi zinginezo isipokuwa Kenya?
Ndio.......La.......
a. Kama ndio, we husafiri wapi?
b. Je, mwisho utembelea nchi gani?.................lini?...........
4. Je, wewe husafiri mara nyingi na Kenya nje ya unapoishi sasa? Ndio.......
La.......
a. Kama ndio, wapi?
b. Nchi gani ama sehemu gani ulisafiria mwisho?.................lini?...........
5. Umeoa/Umeolewa? Ndio....... La....... (kama La, endelea na #6)
a. Kama uko ndoani, je, kwa sasa unaishi pamoja na wenzi wako?
Ndio....La....
i. Je, wewe uhusiano wako na mwenzio ni ya ubinafsi?
Ndio.......La.......
b. Kama si pamoja, huwa mnawahi kuwasiliana kimwili? Ndio........La......
i. Mara ngapi mna jiunga ama wasiliana kwa ubinafsi:
ii. Je, ni muungano wa karibu wa kindoa?:
6. Je, una mpenzi/ wapenzi wa kindoa? Ndio..... .La.....
a. Kama ndio, wangapi?............

89
b. We waja nao pamoja kwa ubinafsi wa ndoa? Ndio..... La.....
c. Kama ndio, mara ngapi?...................
7. Ume wahi kupimwa viini vya Ukimwi? Ndio..... .La.....Kama la, unakubali
kupimwa?
8. Unajua hali yako kidamu ya ukimwi? Ndio..... .La..... Kama la, wataka kujua?
9. Je, Mwenzi/Mpenzi wako ajua hali yako ya ukimwi? Ndio..... La.....
a. Kama la, unataka/unakubali wajulishwe? Ndio..... .La.....
b. Kama ndio, unahitaji ushaidishi kuwajilisha hali yako? ndio..... .la.....
10. Je, Mpenzi wako amewahi kupimwa kwa ajili ya Ukimwi? Ndio.. La....
a. Kama ndio, unajua hali yake/yao? Ndio..... La.....
b. Kama la, wewe uendelea kuwa naye/nao pamoja kimwili? Yes.... No....
11. Ni mda gani sasa tangu ulijua kuhusu hali yako ya Ukimwa mara ya kwanza?
12. Je, unatumia dawa ya kutibi Ukimwi? Ndio......La.....
a. Kama ndio, kwa mda gani sasa tangu uanze kuzitumia?
b. Je, umewahi kubadilishiwa madawa haya kwa ajili yeyote?
i. Kama ndiyo, mara ngapi umebadili madawa haya?
c. Je, umewahi kusimamisha utumishi wa madawa haya kwa sababu
yoyote?
d. Umewahi kusahau ama kuahirishwa uimishi wa haya madawa popotepo?
i. Mara ngapi?
e. Je, kwa sasa unatumia dawa hizo mara kwa mara? Ndio..... La.....
13. Je, kama hutimii dawa ya Ukimwi, unajua waweza kuzipata bure? Ndio......La.....
a. Je, unataka kuanza kutumia dawa ya kutibu Ukimwi? Ndio......La.....
14. Je, unakubaliana na kuhifadhi kwa damu/sampuli yako kwa ajili ya utumizi wa
baadaye?
15. Je, kuna kitu unataka kuniambia au maswali unataka kuuliza?

90
Appendix 4: Analysis of concordance between various subtyping tools
a) Analysis for the Envelope C2V3 Region
Patient NCBI conco COMET conco REGA conco jpHMM conco Phylogen
ID AMP rd rd rd rd
HND046 A1 Y A1 Y A1 Y A1 Y A1
HND164 D/CRF10 P D Y D Y D Y D
HND177 A1 Y A1 Y A1 Y A1 Y A1
KAH001 A2/C P A2 (check P N/A N A2 Y A2
for
02_AG)
KAH004 A1/CRF0 P A1 Y A1 Y A1 Y A1
2
KAH010 A1 Y unassigne N N/A N A1D P A1?
d_1;45_cp
x, D
KAH020 CRF07/C P C Y N/A N C Y C
KAH029 A1/A2 P A1 Y A1 Y A1 Y A1
KAH090 C/CRF07 P C Y C Y C Y C
KAH138 D Y D Y D Y D Y D
KAH139 A1/CRF0 P A1 (check P N/A N A1 Y A1
2 for
02_AG)
KAH142 A1/C P A1 Y N/A N A1 Y A1
KAH144 D/CRF10 P D Y D Y D Y D
KAH148 A1 Y A1 Y A1 Y A1 Y A1
KAH157 A1/U P A1 (check P A1 Y A1 Y A1
for
02_AG)
KAH227 CRF11/C P A1 Y N/A N A1 Y A1
RF02/A1
KAH008 A1 Y A1 Y A1 Y A1 Y A1
KAH009 CRF02/A P A1 Y A1 Y A1 Y A1
1/CRF11
KHC013 A1/CRF1 P A1 Y N/A N A1 Y A1
1/C
KHC016 A1/CRF1 P A1 Y A1 Y A1 Y A1-UG
1
KHC039 A1/CRF1 P A1 Y A1 Y A1A2 P A1
1
KHC040 C/CRF08 P C Y C Y C Y C
KHC045 A1/CRF1 P A1 Y A1 Y A1H P A1
1
KHC048 A1/CRF0 P A1 Y A1 Y A1A2 P A1

91
Patient NCBI conco COMET conco REGA conco jpHMM conco Phylogen
ID AMP rd rd rd rd
2
KHC050 A1/CRF0 P A1 Y A1 Y A1 Y A1
2/CRF11 (100)
KHC052 C/CRF08 P C Y C Y C Y C
KHC058 A1 Y A1 Y A1 Y A1 Y A1
KHC059 A1/CRF1 P A1 Y A1 Y A1 Y A1
1
KHC064 A1/CRF1 P unassigne N N/A N A1D P A1
0/CRF13/ d_2;02_A
CRF02/ G, 10_CD
KHC069 A1/CRF0 P A1 Y N/A N A1 Y A1
2/CRF11
KHC160 A1 Y A1 Y A1 Y A1 Y A1
KHC163 A1/CRF0 P A1 Y A1 Y A1 Y A1
2
KMB031 CRF02/A P A1 Y A1 Y A1 Y A1-UG
1
KMB039 CRF02/B N A1 (check P A1 Y A1A2 P A1-UG
for
02_AG)
KMB067 A1 Y A1 (check P A1 Y A1 Y A1
for
02_AG)
KMB154 A1 Y A1 Y N/A N A1 Y A1
MLD011 A1/D P unassigne P N/A N A1D P A1?
d_1;D,
A1,
02_AG
MLD012 A1/C P unassigne P N/A N A1A2D P A1?
d_1;D, A1
MLD016 A1/A2 P A1 Y N/A N A1 Y A1?
MLD185 A1 Y A1 Y A1 Y A1 Y A1
MLD245 CRF16/A P A1 N N/A N A2 Y A2
2
MLD258 A1/CRF1 P A1 Y A1 Y A1 Y A1
1
MLD301 CRF02 N A1 Y A1 Y A1 Y A1
NVS008 D Y D Y D Y D Y D
NVS032 A1 Y A1 Y A1 Y A1H P A1-UG
NVS040 A1/CRF0 P A1 Y A1 Y A1 Y A1-UG
2
NVS051 A1 Y A1 Y A1 Y A1 Y A1
NVS056 A1 Y A1 Y A1 Y A1 Y A1-UG

92
Patient NCBI conco COMET conco REGA conco jpHMM conco Phylogen
ID AMP rd rd rd rd
NVS074 A2/CRF1 P A1 N A1 N A2 Y A2
6
NVS085 A1 Y A1 Y A1 Y A1 Y A1?
NVS090 A1/CRF1 P A1 Y A1 Y A1 Y A1?
1
NVS095 A1/A2 P A1 Y A1 Y A1 Y A1
NVS108 A1/G P A1 Y A1 Y A1 Y A1-UG
NVS121 A2/CRF1 P A2 Y N/A N A2 Y A2
3
NVS132 CRF02/C N A1 (check P A1 Y A1 Y A1-UG
RF13 for
02_AG)
NVS135 A1/CRF1 P A1 Y A1 Y A1 Y A1?
1
NVS148 CRF07/C P C Y C Y C Y C
RF08/C
NVS152 D Y D Y D Y D Y D
Summary of Concordance
Y-17 Y- 46 Y- 41 Y-49
P- 38 P-8 P-0 P-9
N- 3 N -4 N-
17

93
b) Analysis of concordance for the Pol RT region
Patient NCBI C Stanford C REGA C jpHMM C G2P C COMET C Squeal C Phylogen
ID
HND200 A1,CRF01 P CRF01_A P A1 (96) Y A1 Y A1 Y A1 Y A1 Y A1-KE
E (94.4) (100%)
KAH004 CRF10/A1 N D (90.4) P N/A Y A1D N 10_CD N unassigned Y Complex Y Unassigned
/U/CRF16 (100) _2;02_AG,
10_CD
KAH010 A1/CRF01 P CRF01_A P A1 (97) Y A1 Y A1 Y A1 Y A-ancestral P A1-KE
/CRF15 E (92.6) (100%)
KAH016 A1/CRF01 P CRF01_A P A1 (91) Y A1C P A1 Y unassigned P Complex N A1-KE
/CRF15 E (93.4) (100%) _1;C, A1
KHC016 A1/CRF15 P CRF01_A N A1 (93) N A1 N A1 N A1 N A-ancestral N G or AG
/G E (93.1) (100%)
KHC025 D/CRF10 P D (94.2) Y D (73) Y D Y D (100) Y D Y A4,D P D
recombinan
t
KHC040 C Y C (93.2) Y C (100) Y C Y C (100) Y C Y C Y C
KHC045 A1/CRF15 P CRF01_A P A1 (98) Y A1 Y A1 Y A1 Y Complex N A1-UG
/CRF01 E (92.5) (100%)
KHC048 A1 Y A (92.5) Y A1 (97) Y A1 Y A1 Y A1 Y A1 Y A1-KE
(100%)
KHC106 A1/CRF01 P CRF01_A P A1 Y A1 Y A1 Y A1 Y A1 Y A1-KE
/CRF15 E (93.2)
KHC160 A1 P CRF01_A P A1 (99) Y A1 Y A1 Y A1 Y A1 Y A1-KE
E (93.3) (100%)
KHC163 A1/CRF01 P CRF01_A P A1 (100) Y A1 Y A1 Y A1 Y A1 Y A1-KE
/CRF16 E (93.1) (100%)
KHC093 A1 Y A (93.3) Y A1 (100) Y A1 Y A1 Y A1 Y A1 Y A1-KE
(100)
KMB039 A2/A1/CR P CRF01_A N A1 (94) N A1A2 P A2 Y unassigned P A,A2 P A2
F16 E (92.3) (100%) _1;A2, A1 recombinan
t
KMB067 A1/CRF01 P A (92.5) Y A1 (98) Y A1 Y A1 Y A1 Y A1 Y A1-UG
/CRF15 (100%)
KMB145 A1/CRF15 P CRF01_A P A1 (97) Y A1 Y A1 Y A1 Y A1 Y A1-KE
E (93.7) (100%)
KMB160 A1/CRF15 P CRF01_A P A1 (98) Y A1 Y A1 Y A1 Y A1 Y A1-UG
/CRF02 E (91.6) (100%)
KMB077 CRF10/A1 N D (92.4) N NA N D N 10_CD P unassigned N Complex Y C?
/CRF15 _1;39_BF,
A1
MLD011 D/CRF10 P D (94.9) P D (100) P D P 10_CD Y D P D P 10_CD
MLD183 A1/H/CRF P A (91.6) Y N/A N A1/H P A1 Y unassigned P A1,D P A1-KE
05 (100) _1;H, A1 recombinan
t
MLD185 A1/CRF15 P CRF01_A P A1 (93) Y A1 Y A1 Y A1 Y A1 Y A1-KE
/CRF02 E (93.7) (100%)
MLD191 D/CRF10 P D (94.3) Y D (89) Y D Y D (100) Y D Y D Y D
MLD198 A1/CRF01 P A (93.7) Y A1 (100) Y A1 Y A1 Y A1 Y A-ancestral Y A1-KE
MLD021 CRF10/D/ P D (100) Y D (100) Y D/K/D P 10_CD P D Y A1,D P D
CRF05/B recombinan

94
Patient NCBI C Stanford C REGA C jpHMM C G2P C COMET C Squeal C Phylogen
ID
t
MLD245 CRF16/A2 P B (91.4) N N/A N A2/B P A2 P B (check for P CRF16-like P A2?
/B (100%) 16_A2D)
MLD040 A1/CRF01 P CRF01_A N N/A N C/A1 P A1 Y unassigned P A1,C P A1-KE
/A2/C E (93.5) (100) _1;C, A1 recombinan
t
MLD541 CRF15/CR P CRF01_A Y N/A N A1/K N CRF01_ Y A1 P A-ancestral N 01_AE
F01/U E (92.4) AE
MLD545 A1 Y A (93.0) Y A1 (100) Y A1 Y A1 Y A1 Y A1 Y A1-KE
(100)
MLD548 D/CRF10/ N D (93.8) N N/A Y D N D (100) N unassigned Y U Y Unassigned
A1 _1;D, A1
MLD060 C Y C (94.1) Y C (100) Y C Y C (100) Y C Y C Y C
NVS032 A1/CRF15 P CRF01_A P A1 (100) Y A1 Y A1 Y A1 Y A,A1 P A1-KE
/CRF01 E (92.2) (100%) recombinan
t
NVS047 A1/CRF01 P CRF01_A P A1 (98) Y A1 Y A1 Y A1 Y A1 Y A1-KE
E (92.3) (100%)
NVS052 A1/CRF01 P A (93.2) Y A1 (94) Y A1 Y A1 Y A1 Y Complex N A1-KE
/C (100%)
NVS056 A1 Y A (93.4) Y A1 (99) Y A1 Y A1 Y A1 Y A1 Y A1-KE
(100%)
NVS069 A1/CRF01 P CRF01_A P A1 (87) Y A1 Y A1 Y A1 Y A1 Y A1-KE
/CRF15 E (91.7) (100%)
NVS085 A1/CRF01 P A (93.4) Y A1 (100) Y A1 Y A1 Y A1 Y A,A1 P A1-UG
/CRF15 (100%) recombinan
t
NVS090 A1/CRF01 P A (92.8) Y A1 (97) Y A1 Y A1 Y A1 Y A1 Y A1-KE
/CRF15
NVS095 A1/CRF01 P A (91.9) Y A1 (98) Y A1 Y A1 Y A1 Y A,A1 P A1-KE
(100) recombinan
t
NVS122 A1 Y A (92.8) Y A1 (98) Y A1 Y A1 Y A1 Y A1 Y A1-KE
(100%)
NVS152 D/CRF10/ P D (91) N N/A N D N 10_CD P C Y C,D P C
C (100) recombinan
t
NVS058 A1/CRF01 P A (92.3) Y NA N A1/C P A1 Y unassigned P Complex N A1-UG
/CRF09 _1;C, A1

Summary of concordance
Y–7 Y – 19 Y - 31 Y – 27 Y – 34 Y – 31 Y – 23
P – 31 P – 15 P–1 P–8 P–4 P–8 P – 12
N-3 N-7 N-9 N-6 N-3 N-2 N-6

95
Appendix 5: Coreceptor Usage and Number of Potential N-Glycosites
Patient ID Coreceptor Total PNGs NXT NXS NNXST
1 HND046 CCR5 12 8 4 0
2 HND164 CXCR4 10 7 3 0
3 HND177 CCR5 8 7 1 0
4 KAH001 CCR5 12 8 4 0
5 KAH004 CCR5 6 4 2 0
6 KAH008 CCR5 12 7 5 0
7 KAH009 CXCR4 13 11 2 0
8 KAH010 CCR5 10 7 2 1
9 KAH020 CCR5 11 6 5 0
10 KAH029 CCR5 14 10 4 0
11 KAH090 CCR5 9 6 3 0
12 KAH138 CXCR4 12 9 3 0
13 KAH139 CXCR4 13 8 5 0
14 KAH142 CCR5 13 9 3 1
15 KAH144 CCR5 14 9 4 1
16 KAH148 CCR5 12 9 3 0
17 KAH157 CCR5 14 9 5 0
18 KAH227 CXCR4 12 8 4 0
19 KHC013 CCR5 13 8 5 0
20 KHC016 CCR5 11 6 5 0
21 KHC039 CCR5 8 5 3 0
22 KHC040 CCR5 12 7 5 0
23 KHC045 CCR5 6 5 1 0
24 KHC048 CXCR4 10 6 4 0
25 KHC050 CCR5 13 10 3 0
26 KHC052 CCR5 11 9 2 0

96
Patient ID Coreceptor Total PNGs NXT NXS NNXST
27 KHC058 CCR5 11 7 4 0
28 KHC059 CCR5 10 4 6 0
29 KHC064 CCR5 14 8 6 0
30 KHC069 CXCR4 10 5 5 0
31 KHC160 CCR5 8 4 2 2
32 KHC163 CCR5 10 5 4 1
33 KMB031 CCR5 7 4 3 0
34 KMB039 CXCR4 7 6 0 1
35 KMB067 CCR5 11 6 4 1
36 MLD011 CCR5 11 7 4 0
37 MLD012 CCR5 11 7 4 0
38 MLD016 CCR5 9 7 2 0
39 MLD185 CCR5 13 8 5 0
40 MLD245 CXCR4 11 6 5 0
41 MLD258 CCR5 12 7 5 0
42 MLD301 CCR5 7 4 3 0
43 NVS008 CXCR4 12 8 4 0
44 NVS032 CCR5 10 7 3 0
45 NVS040 CCR5 13 9 4 0
46 NVS051 CXCR4 13 7 6 0
47 NVS056 CCR5 13 9 4 0
48 NVS074 CCR5 11 10 1 0
49 NVS085 CCR5 8 5 3 0
50 NVS090 CCR5 12 10 2 0
51 NVS095 CXCR4 13 9 4 0
52 NVS108 CCR5 14 9 5 0

97
Patient ID Coreceptor Total PNGs NXT NXS NNXST
53 NVS121 CXCR4 9 7 2 0
54 NVS132 CCR5 11 7 4 0
55 NVS135 CCR5 9 6 3 0
56 NVS148 CCR5 13 9 4 0
57 NVS152 CXCR4 14 9 5 0
Total 636 417 210 9

98
Appendix 6: Request for SSC and ERC waiver
THE DIRECTOR,
ITROMID, JKUAT,
P.O. BOX 62000-00200,
NAIROBI, KENYA.
3/2/2014
Dear Sir/Madam,
RE: MSc RESEARCH PROJECT FOR ROSE CHARI KITAWI; STUDENT
REGISTRATION TM 306-0957/2012
The above named MSc. Medical Epidemiology Student is one of the students that I
enlisted to complete her thesis project under my guidance. Rose‟s project is hived out of
a larger study, SSC #2477, “Genetic Characterization of HIV-1 Strains and
Determination of Known Mutations Conferring Resistance to Antiretroviral Drugs in
Kenya”. Both the Scientific Ethical committees of KEMRI already approve SSC #2477.
Rose has started working on her part of the study, which is focused on generating the
below data from Kisumu, Nakuru and Kajiado counties;

1. To determine circulating HIV strains and their genetic diversity in Kisumu, Nakuru
and Kajiado Counties
2. To determine the patterns of HIV co-receptor usage in Kisumu, Kajiado and
Nakuru Counties
3. To compare trends of HIV-1 and deduce circulating patterns (dispersal,
transmission, spread) of the virus from Kisumu, Nakuru and Kajiado Counties.
4. To assess HIV-1 envelope diversity based on the patterns of Potential N-
Glycosylation sites of different viral genotypes.
Please consider waiving any requirement that Rose‟s study be individually approved by
the SSC and ERC based on the existing approval (enclosed) of the parent study to which
hers is appended.
Sincerely

Washingtone Ochieng’, Ph.D,


Centre for Virus Research, KEMRI

99
Appendix 7: ERC Approval

100
Appendix 8: SSC Approval

101

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